Network pharmacology and in vivo investigation of the growth and immunity-enhancing mechanisms of Tetramethylpyrazine (TMP) in Litopenaeus vannamei.

In this study, the shrimp (L. vannamei, 0.89 ± 0.04 g) were fed formulated diets supplemented with Tetramethylpyrazine for six weeks under routine aquaculture conditions, followed by a three-day challenge with White Spot Syndrome Virus (WSSV). The results showed that dietary TMP supplementation significantly improved the specific growth rate and weight gain rate, while reducing the feed conversion ratio. During the viral challenge, the immune protection rate of shrimp was significantly enhanced (P < 0.05). Further analysis using network pharmacology, molecular docking, and quantitative real-time PCR demonstrated that TMP exhibited strong binding affinities with hypoxia-inducible factor 1-alpha (HIF1A), prostaglandin synthase (PTGSH2), and nuclear transcription factors (Relish and Dorsal). At the transcriptional level, TMP significantly inhibited the expression of HIF1A and sterol regulatory element-binding protein (SREBP), while upregulating adiponectin receptor (AdipoR) expression. TMP also promoted lipid catabolismand cholesterol efflux-related genes, while downregulating lipid synthesis genes. Moreover, TMP enhanced the expression of arachidonic acid pathway-related genes and antioxidant enzymes, leading to reduced levels of malondialdehyde (MDA) and blood lipids. Regarding immune stimulation, TMP activated the NF-κB (Dorsal, Relish) signaling pathway, upregulated the expression of antimicrobial peptides, and reduced inflammatory cytokine levels. Under WSSV infection, TMP suppressed viral replication by downregulating HIF1A and VEGF expression (P < 0.05), suggesting that TMP exerts its antiviral effect. High-throughput sequencing of the intestinal microbiota indicated that TMP supplementation increased the abundance of beneficial bacteria and decreased the abundance of harmful bacteria, thereby improving intestinal mucosal barrier function. In conclusion, considering both growth performance and physiological health, we recommend an optimal TMP supplementation level of 150 mg/kg in shrimp feed.