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

Withania somnifera (L.) Dunal (WS), also known as ‘Ashwagandha’, has been used to treat a variety of diseases such as arthritis, anxiety, and insomnia in Human. However, there is limited report on the application of dietary WS in aquaculture. This study was aimed at demonstrating the stress-alleviation and hepatoprotective mechanisms of dietary WS supplementation in female adult darkbarbel catfish. Adult female darkbarbel catfish were fed with WS dietary for 5 weeks, and treated by physical stimuli (injection of normal saline). Then the brain tissues were collected for transcriptome profile analysis (RNA-seq) to identify DEGs associated with stress response. Serum samples were collected for analysis of related biochemical indices associated with stress and immune responses. Liver tissues were collected for analysis of lipid accumulation and cell proliferation through Oil Red O staining and PCNA immunostaining. Moreover, the network pharmacological analysis was conducted: The bioactive compounds of WS were screened and their potential targets predicted by searching the ChEBI and PCIDB databases, then potential targets were predicted using the Swiss Target Prediction database, thus the “WS-ingredient-target” regulatory network was constructed using Cytoscape software. The binding affinities of key active compounds from WS to target proteins were analyzed by molecular docking. Then Data were validated in young darkbarbel catfish, and in SG3 cells treated by Withanolide A. The comparative transcriptomic analysis of brain tissues revealed that physical stimulus strongly induced the expression of genes related to oxidative stress and immune response (1451 DEGs), while this stress response was markedly mitigated in WS-supplemented fish (only 397 DEGs). The 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 young darkbarbel catfish and enhanced proliferation potency of SG3 cells. Intriguingly, in the adult female darkbarbel catfish, it was found that dietary WS could significantly reduce the levels of cortisol, glucose, ROS, TNF-α, and IL-6 in fish serum, improve the PCNA expression in hepatocytes and allievate the liver injuries caused by physical stress (p < 0.05). 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. Also, the findings would provide novel index for further extensive investigations on the targets and applications of traditional Chinese medicine (TCM) in fish species in the future.