ZEBRAFISH PLATFORM FOR ANTI-INFLAMMATORY DRUG DISCOVERY

Tomato and Olive Bioactive Compounds (TOBC) are a patented nutraceutical formulation derived from tomato and olive mill wastewater extracts. In this study, through integrated in vivo and in vitro approaches, we demonstrate the strong ability of TOBC to promote tissue regeneration by finely modulating early and late inflammatory responses. In the in vivo zebrafish model, caudal fin amputation was performed at 72 hours post fertilization (hpf), and larvae were treated with TOBC at 1 or 2 mg/mL. Using the Tg(mpx:GFP) transgenic line, we evaluated how these concentrations influenced neutrophil behaviour following injury. Neutrophil counts, quantitative fluorescence imaging and RT-PCR analyses revealed that TOBC at 1 mg/mL enhanced neutrophil recruitment and increased interleukin 1 beta (il1β) and interferon regulatory factor 8 (irf8) expression at 6 hours post amputation (hpa), along with an early rise of the active Il1β form at 2 hpa, indicating an amplified early inflammatory activation. We further examined TOBC during later stages of epimorphic regeneration (24 and 48 hpa). TOBC promoted a pro-regenerative environment by modulating macrophage recruitment to the wound site and increasing both tumor necrosis factor alpha (tnfa) mRNA expression together with Tnfa protein levels at 24 hpa. At 48 hpa, TOBC enhanced proliferative activity and fin regrowth, as demonstrated by elevated proliferating cell nuclear antigen (PCNA) and transforming growth factor beta 1 (Tgfβ1) protein levels. Co-treatment with the caspase-1 inhibitor Ac-YVAD-CMK (N-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone) completely abolished TOBC-induced effects, indicating that its regenerative activity strictly depends on caspase-1 mediated Il1β maturation and inflammasome activation. These in vivo findings were complemented by in vitro experiments performed using the human bronchial epithelial BEAS-2B cell line, a widely used and well-established non-tumorigenic epithelial model for studying inflammatory and oxidative stress responses. Human bronchial epithelial cells play a key role in orchestrating inflammatory signalling and tissue homeostasis through the release of cytokines, growth factors and redox mediators, processes that are critically involved in inflammation-driven tissue repair. For this reason, BEAS-2B cells were selected as a human epithelial model to assess whether TOBC-mediated immunomodulatory and pro-regenerative effects observed in vivo are conserved at the epithelial level in a mammalian system. In this context, TOBC at 1 mg/mL increased cell viability and proliferation, as confirmed by accelerated progression through the S and G2/M phases of the cell cycle. Scratch assays revealed faster wound closure in TOBC-treated cultures, and TOBC additionally stimulated IL-1β and epidermal growth factor (EGF) secretion, supporting the establishment of a pro-regenerative microenvironment. In conclusion, the use of BEAS-2B cells provided complementary mechanistic insight into conserved epithelial responses relevant to inflammation-driven tissue repair and airway remodelling, thereby strengthening the translational relevance of the zebrafish model. Together, these results identify TOBC as a promising candidate for therapeutic applications in tissue repair and inflammation control. Its combined ability to modulate Tnfα-dependent inflammatory pathways, rely on caspase-1 mediated Il1β activation, and directly stimulate cellular proliferation and remodelling highlights its dual regenerative and immunoregulatory potential.