Unraveling the Dual Threat: How Polystyrene Nanoplastics and Benzo(a)pyrene Synergistically Induce Lung Fibrosis and Inflammation via Relaxin Signaling

Micro- and nanoplastics (MNPs) are emerging pollutants that can ferry persistent organic pollutants such as benzo(a)pyrene (BaP). While pulmonary toxicity of nanoplastics (NPs) is documented, how combined NP and BaP exposure triggers cellular crosstalk remains elusive. We established a 16-week mouse model receiving environmentally relevant doses of polystyrene NPs, BaP, or NPs+BaP (Mix). Histopathology showed extensive inflammatory infiltration and marked fibrotic lesions only in Mix lungs; single exposures had minimal effects. Mix animals displayed sharply elevated oxidative stress indices (MDA) alongside depleted antioxidants (GSH, CAT, T-AOC, SOD). Expression of inflammatory cytokines IL-1b and TNF-a and the fibrotic marker a-SMA rose, whereas E-cadherin fell, confirming synergistic toxicity. Transcriptomic profiling revealed significant enrichment of the Relaxin signalling pathway. Mechanistically, Relaxin1 and Relaxin3 modulated inflammation- and fibrosis-related genes via PI3K-AKT and MAPK cascades; Relaxin4 activated PLC-IP3R, opening ER calcium channels and raising cytosolic Ca2+, which triggered macrophage extracellular trap (MET) formation. ER Ca2+ release proved essential for METs, and calcium chelation blocked MET induction by combined exposure. A macrophage-MLE-12 co-culture system further verified that Mix-induced METs drove alveolar inflammation and fibrosis. Our findings unveil novel molecular circuits linking environmentally relevant co-exposure to PS-NPs and BaP with exacerbated lung fibrosis and inflammation, highlighting Relaxin-Ca2+-MET signalling as a potential intervention node.