Integration of multi-omics and network toxicology reveals TLR4-mediated nephrotoxicity induced by arecoline

Arecoline, the primary alkaloid in areca nut, induces renal injury, yet its underlying molecular mechanisms remain poorly understood. This study aimed to identify arecoline’s molecular targets and elucidate the subsequent pathological signaling cascade in renal cells. We integrated network toxicology, molecular docking, and molecular dynamics (MD) simulations to predict arecoline’s targets. In vitro validation in human kidney (HK-2) cells assessed target engagement via a cellular thermal shift assay (CETSA). Downstream signaling, inflammatory, and fibrotic markers were evaluated using Western blotting, ELISA, and immunofluorescence. Computational models predicted a stable arecoline-Toll-like receptor 4 (TLR4) complex. CETSA demonstrated that arecoline enhanced TLR4 thermal stability in HK-2 cells, strongly indicating target engagement. This binding activated the PI3K/AKT/NF-κB signaling pathway. Consequently, arecoline induced NF-κB-dependent upregulation of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), elevated the renal injury marker KIM-1, and promoted the accumulation of fibrotic proteins (α-SMA, Collagen III). Arecoline instigates nephrotoxicity by targeting TLR4 and activating the PI3K/AKT/NF-κB axis, driving an inflammatory and fibrotic phenotype in renal tubular cells. The arecoline-TLR4 interaction represents a critical event in areca nut-associated nephropathy, offering a novel therapeutic target.