Network Pharmacology and In Vitro Experimental Validation Reveal the Anti-Inflammatory and Anti-Apoptotic Effects of Lotus Leaf Extract in Treating Inflammatory Diarrhea in Pigs.

The objective of this research was to investigate the efficacy of lotus leaf in the prevention and treatment of inflammatory diarrhea in pigs, utilizing network pharmacology and in vitro methodologies. Initially, LC-MS was employed to analyze the constituents of lotus leaf extract (LLE); then, the TCMSP database was utilized to identify the active components and their corresponding targets. The GeneCards database was consulted to identify disease-related targets pertinent to inflammatory diarrhea in pigs. A drug ingredient-disease target network was constructed using Cytoscape software. Subsequently, the STRING database facilitated protein interaction analysis, which was also visualized through Cytoscape. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted based on the genes shared between disease and LLE targets. Molecular docking of the active ingredients with key targets was performed using Autodock Vina. Subsequently, an in vitro LPS-induced inflammation model was established using IPEC-J2 cells to validate the predictions made through network pharmacology. Verification was conducted via flow cytometry and Western blot analysis. The LC-MS assay and TCMSP retrieval results revealed that Quercetin, Nuciferine, Kaempferol, Leucodelphinidin, and Catechin were identified as the main compounds of LLE, associated with 181 potential targets. A total of 5995 targets were linked to inflammatory diarrhea in pigs, with 159 overlapping targets identified between the bioactive compounds and the disease. Notable key targets included TNF-α, IL-6, caspase-3, TP53, and AKT, which are integral to inflammation and apoptosis processes. GO functional annotation indicated significant enrichment in biological processes such as gene expression regulation and transcription from RNA polymerase II promoters. KEGG pathway analysis highlighted critical pathways, including TNF signaling and apoptosis. Furthermore, molecular docking analyses demonstrated that the bioactive components of lotus leaf exhibited a strong binding affinity for essential targets, including AKT1, BAX, caspase-3, CCL2, IL-6, IL-10, MPK14, NOS3, PTGS1, and TNF-α. In vitro experiments confirmed that LLE significantly inhibited LPS-induced apoptosis in IPEC-J2 cells and suppressed the activation of the TNF-α-mediated apoptosis pathway. This study offers novel insights into the therapeutic potential of Chinese medicine and its constituents in addressing inflammatory diarrhea in pigs.