Effects and mechanisms of dietary Osthole (OST) and Tetramethylpyrazine (TMP) on growth performance, antioxidant capability and immune response of Pacific white shrimp ( <i>Litopenaeus vannamei</i> )

Dietary plant-derived bioactive compounds for enhancing physiological health are becoming a prevalent strategy for antibiotic alternatives. Our study revealed the effects and underlying mechanism of Osthole (OST) and OST-Tetramethylpyrazine (TMP) compound in <i>Litopenaeus vannamei</i> based on network pharmacology, molecular docking and a 42-day feeding trial verification. The results illustrated that OST and OST-TMP compound significantly improved the survival rate (SR), weight gain rate (WGR), specific growth rate (SGR) and feed conversion ratio (FCR), strengthening the growth performance of <i>L. vannamei</i>. Meanwhile, combining the predictive results from network pharmacology and molecular docking, we propose that OST and TMP synergistically enhance the antioxidant defense capacity of shrimp through the synergistic Nrf2 signaling pathway, thereby enhancing the expression of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Furthermore, OST-TMP exhibited a significant increase of the immune response in hemocyte and intestine of shrimp, increasing the expression of antimicrobial peptide and lysozyme, and suppressing the inflammatory factors, via the synergistic (NF-κB) and complementary targets predicted by network pharmacology. Additionally, gut microbiota composition of <i>L. vannamei</i> was improved, and the dominant genera were correlated with intestinal immune in the compound groups. For the first time, we elucidated the mechanism of plant-derived bioactive compounds mediating physiological health in aquatic animals via a new strategy of network pharmacology-molecular docking-experimental verification, and identified the optimal addition amount of OST-TMP in shrimp (150 mg/kg TMP + 20 mg/kg OST), providing a technical safeguard for the animal health and the safety of aquatic products.