Disruption of pulmonary surfactant function under oxidative stress: structural and biophysical insights

Abstract

Pulmonary surfactant forms surface-active films at the air-liquid interface which are essential for alveolar stability but are highly susceptible to oxidative damage under inflammatory conditions. The reactive species hypochlorous acid (HClO/ClO−), generated by activated neutrophils, is a potent oxidant that may compromise surfactant integrity; however, its molecular and functional impact remains incompletely understood. In this study, the effects of oxidative stress induced by HClO/ClO− on the biophysical and structural properties of a fully native porcine surfactant were investigated using Langmuir balance, captive bubble surfactometry, differential scanning calorimetry, FTIR-ATR spectroscopy, and epifluorescence microscopy. Oxidation induced concentration- and time-dependent inhibition of surfactant adsorption and interfacial film stability. Langmuir balance experiments revealed selective disruption of films containing unsaturated phospholipids or native mixtures, while saturated monolayers remained largely unaffected. FTIR-ATR analysis showed perturbations in lipid headgroup regions and protein secondary structure rearrangements towards β-sheet-rich aggregates, consistent with oxidative unfolding and aggregation. These molecular changes compromise protein-lipid interactions, delaying adsorption and respreading at the air-liquid interface. Epifluorescence microscopy further revealed disruption of lateral lipid organization and altered formation of three-dimensional surfactant reservoirs enriched in disordered components. Functional assays confirmed impaired reduction of surface tension during quasi-static cycling, partially alleviated under dynamic cycling, suggesting exclusion or depuration of oxidized components from the interface. Together, these findings demonstrate that neutrophil-derived oxidants progressively compromise pulmonary surfactant by targeting lipid-protein cooperativity, leading to early interfacial dysfunction followed by structural reorganization. This mechanistic insight advances understanding of oxidative surfactant inactivation and supports the development of antioxidant strategies or oxidant-resistant surfactant formulations as potential therapeutic interventions for inflammatory lung injury.