## 3 Discussion
These in vitro tests provide early and specific intestinal toxicity assays for identifying dietary compounds that may affect intestinal barrier function and represent a valuable tool for further characterizing oregano EO safety in humans, as already shown with oral toxicity tests performed on rats [47]. Data reported in the literature relating to oregano EO anti-inflammatory activity suggest their ability to reduce inflammation in several in vitro and in vivo models [48; 49; 50]; however, in our intestinal Caco-2 cell system, pre-incubation with EOs did not induce evident protection from TNF-\(\alpha\) inflammatory challenge. Nevertheless, CAR1 preincubation followed by TNF-\(\alpha\) exposure determined a weak downregulation of genes involved in the NF-kB pathway. This trend might confirm potential CAR1 anti-inflammatory activity in this experimental system, which could not be further assayed at a higher CAR1 concentration due to its toxicity. Finally, neither CAR1 nor THY5 displayed inflammatory action on their own, as incubation with these EOs did not induce any significant increase in the expression of the inflammatory genes tested, as compared to the control cells. As reported by [PERSON] et al. [31], the initial stages of pathogen adhesion to intestinal cells are crucial for the establishment of colonization, with the risk of being a reservoir of acute events; thus, the search for new inhibitors of bacterial adhesion to host tissues turns out to be very important. The results of the pathogen adhesion assay, described in the present work, showed that the addition of CAR1 and THY5 oils, despite being at low concentrations, was indeed able to reduce bacterial adhesion to the intestinal Caco-2 cell line, although THY5 was effective only against \(S\). Typhimurium LT2. Our data are comparable to those of other research carried out on thyme EOs, in which both thymol (about 12%) and carvacrol (about 68%) were present; in particular, it was observed that both _Thymus capitatus_ EO as well as thymol and carvacrol alone were able to reduce the adhesion of \(E\). _coli_ (ATCC35210) and \(L\). _monocytogenes_ (NCTC 7973) to the intestinal HT-29 cell line [51]. Previous work has demonstrated that carvacrol was able to reduce _Campylobacter jejuni_ infection in epithelial cells [52], while thymol decreased the adhesion of various \(E\). _coli_ and \(S\). _aureus_ strains on vaginal epithelial cells [53]. Finally, both compounds were able to reduce the adhesion of three \(L\). _monocytogenes_ strains to Caco-2 cells [54]. Furthermore, in a recent study carried out on _Origanum vulgare_ EO, a reduction in the adhesion of several _Salmonella_ serotypes to Caco-2 cells was observed by a commercial mixture of EO containing both thymol and carvacrol; this effect was suggested to occur through the inhibition or disaggregation of bacterial biofilm [31]. The mechanisms underlying this effect can be multiple and ascribable to the antimicrobial activity of EOs against Gram-positive and Gram-negative pathogens, to their ability to alter the chemical-physical properties of the bacterial surface or microbial motility, as well as to the reduction in gene expression of some virulence factors [52; 53; 54]. Further experiments will be needed to fully understand the mechanisms underlying the protective effects exerted by oregano EOs.