Effects of the dietary Withania somnifera on stress-relief and hepatoprotection in darkbarbel catfish (Pelteobagrus vachelli)

Withania somnifera (L.) Dunal (WS), or Ashwagandha, is used clinically against arthritis, anxiety and insomnia etc, however, its potential in aquafeeds is largely unexplored. We investigated the stress-alleviation and hepatoprotective mechanisms of dietary WS supplementation in adult female darkbarbel catfish. Fish were fed with WS dietary for 5 weeks, subjected to a physical stressor (saline injection), and sampled. Brain were processed for RNA-seq to identify differentially expressed genes (DEGs) linked to stress; serum were assayed for stress- and immune-related biochemical markers; livers were examined for lipid content (Oil Red O staining) and proliferation (PCNA immunostaining). Network pharmacology were employed to construct a "compound-target" network: The bioactive compounds of WS were retrieved from ChEBI and PCIDB, probable targets predicted via Swiss Target Prediction, and the “WS-ingredient-target” regulatory network was constructed using Cytoscape; key ligand-protein interactions were validated by molecular docking. Findings were replicated in juvenile fish and in SG3 cells challenged with Withanolide A. Physical stress evoked 1 451 DEGs in control brains, whereas WS-supplemented reduced this to 397, indicating marked mitigation of the oxidative- and immune-stress response. Network pharmacology and molecular docking predicted that 13 bioactive compounds in WS, especially Withanolide A and Withanolide J, can interact with targets such as MAPK8, CRHR1, NR3C2, and IARS1, being associated with stress and liver function regulation respectively. Moreover, it was proved that Withanolide A could significantly modulate stress response in juveniles and enhanced proliferation potency of SG3 cells. Intriguingly, in theadult female darkbarbel catfish, dietary WS lowered serum cortisol, glucose, ROS, TNF-α, and IL-6 ( P < 0.05), up-regulated hepatocyte PCNA expression, and allievated stress-induced liver injury. These findings provide novel insights and vital basis for elucidating the mechanisms whereby herbs, including WS act in stress-relief and hepatoprotection in darkbarbel catfish. Andprovide a framework for advancing traditional Chinese medicine (TCM) applications in aquaculture.