Integrative Systems Pharmacology and Zebrafish Toxicity Profiling Reveal Piperine as a Multi-Target Modulator in...

A bioactive alkaloid derived from Piper nigrum is piperine (PIP); it exhibits significant anti-inflammatory properties, but the mechanistic basis remains incomplete. This study integrates zebrafish toxicity assays, antioxidant profiling and histopathology analysis along with a network pharmacology approach to elucidate system-level understanding of PIP's therapeutic efficacy. Toxicity studies on zebrafish embryos indicated that PIP exhibits low toxicity at elevated dosages (LC₅₀ = 160.25 µg/ml), with preserved hatching and cardiac function. In the DSS-induced intestinal inflammation model, PIP treatment significantly improved swimming behaviour and restored feeding activity. It also preserved lysosomal integrity, reduced apoptosis, and attenuated mucin hypersecretion and ROS (Reactive Oxygen Species) accumulation in a dose-dependent manner. In tissue homogenates, PIP effectively normalized DSS-induced changes in total protein, malondialdehyde (MDA), Glutathione (GSH), Superoxide Dimutase (SOD), catalase and nitric oxide (NO) levels, indicating restoration of redox homeostasis. Furthermore, network pharmacology analysis identified 2,142 PIP-associated targets, of which 55 overlapped with inflammation-related genes. Protein-protein interaction (PPI) network analysis identified key hub genes involved in immune and stress responses. Gene Ontology (GO) enrichment indicated significant involvement in apoptotic signalling, protein phosphorylation, and inflammatory processes, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighted the role of PIP in modulating the PI3K-Akt, JAK-STAT, and MAPK signalling pathways, which are associated with the in vivo inflammatory phenotype. Molecular docking further demonstrated strong binding affinities of PIP with hub proteins, particularly MTOR (- 8.4 kcal/mol) and JAK2 (- 8.3 kcal/mol). Collectively, these findings correlate the observed in vivo phenotypic recovery with multi-target molecular mechanisms, supporting PIP as a promising anti-inflammatory compound for Inflammatory Bowel Diseae (IBD) and related disorders with significant bio-therapeutic potential.