Abstract:
Food-derived lactic acid bacteria (LAB) may contribute to the microbiota–gut–brain axis (MGBA) communication not only through microbial metabolites, but also indirectly through interactions with intestinal epithelial cells and the generation of bioactive soluble factors capable of influencing neuronal function. Therefore, this study aimed to evaluate the neuroactive potential of conditioned medium generated by a defined mixture of food-derived LAB interacting with intestinal epithelial cells, using SH-SY5Y cells as an in vitro neuronal model.
Three food-derived LAB strains, L.paracasei OS4, L. plantarum M3.3, and L.plantarum O24, were combined in equal proportions to prepare a multi-strain LAB mixture (LAB MIX). Caco-2 intestinal epithelial cells were exposed for 24 h to LAB MIX at 10? CFU/mL, and the resulting cell-free conditioned medium (CM-LAB) was subsequently applied to SH-SY5Y neuronal cells. Differentiated SH-SY5Y cells were treated with CM-LAB, and transcriptional responses were assessed by RT-qPCR, focusing on genes related to antioxidant defense (NFE2L2, SOD1, SOD2, and GPX1), apoptosis regulation (BAX and BCL2), and neurotrophic support (BDNF). Cell viability and intracellular ROS levels were also evaluated, and exploratory profiling of the NF-κB pathway-related proteome was performed in SH-SY5Y cells.
CM-LAB generated following Caco-2 exposure to LAB MIX at 10? CFU/mL elicited a distinct transcriptional response in SH-SY5Y cells. Expression of the antioxidant-associated genes NFE2L2, SOD1, SOD2, and GPX1 was significantly increased compared with the conditioned-medium control (p < 0.05). In parallel, BAX expression was reduced, whereas BCL2 and BDNF expression increased, resulting in a markedly lower BAX/BCL2 ratio (p < 0.05). CM-LAB did not induce significant increases or decreases in MTT-derived cell viability or intracellular ROS levels, both of which remained comparable to their respective conditioned-medium controls. Exploratory proteome profiling further indicated selective changes in NF-κB- and stress-related signaling. CM-LAB was associated with higher relative signals of RelA/p65 and its phosphorylated form RelA/p65 [pS529], accompanied by higher IκBα and IκBε signals, suggesting changes in both NF-κB-associated signaling components and their regulatory proteins. In addition, c-Rel, TLR2, JNK2, and cIAP2/BIRC3 were detected only in CM-LAB-treated cells, indicating broader differences in proteins associated with pattern recognition, MAPK-related signaling, and cell survival regulation.
Overall, these findings indicate that soluble factors generated during LAB–intestinal epithelial cell interactions can modulate antioxidant-associated, apoptosis-related, and neurotrophic transcriptional responses in SH-SY5Y cells, together with selective changes in NF-κB- and stress-related protein profiles. Importantly, these molecular responses occurred without significant alterations in cell viability or intracellular ROS levels relative to the conditioned-medium control. In this in vitro epithelial–neuronal model, the results support the potential of food-derived LAB to indirectly influence neuronal responses through soluble factors generated at the intestinal epithelial interface.
