Integrated quantitative proteomics reveals stress-associated network remodeling induced by mitragynine in RSC96...

Mitragynine, the principal alkaloid of Mitragyna speciosa (kratom), exhibits opioid-like analgesic effects but is associated with tolerance following prolonged exposure. Schwann cells are particularly vulnerable to chemically induced toxicity, and disruption of their homeostatic functions has been implicated in neurotoxic injury. The cellular mechanisms underlying this adaptive response remain poorly understood. In this study, an integrated quantitative proteomics and systems biology approach was performed to investigate mitragynine-induced molecular remodeling in RSC96 Schwann cells. Cells were exposed to 20 µM mitragynine, the highest non-cytotoxic concentration, for 72 h and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic profiling. Differentially expressed proteins were characterized through Gene Ontology enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway mapping, InterPro domain annotation, and protein-protein interaction network reconstruction using STRING and BioGRID. Proteomic profiling identified 91 significantly altered proteins, comprising 60 downregulated and 31 upregulated proteins. Functional enrichment revealed coordinated suppression of translational machinery, cytoskeletal organization, and metabolic pathways associated with Schwann cell homeostasis. Network reconstruction highlighted AMP-activated protein kinase (AMPK) as a high-centrality node within the downregulated interaction network. Upregulated proteins were enriched in xenobiotic stress responses, aminoacyl-tRNA biosynthesis, and chromatin remodeling pathways. Structural similarity analysis revealed limited overlap between the mitragynine scaffold and morphine despite their shared receptor target. These findings suggest that chronic mitragynine exposure induces coordinated proteomic and network-level remodeling in Schwann cells, identifying regulatory pathways consistent with tolerance-related cellular adaptation and peripheral neurotoxic risk.