Mechanisms of Anti-Oxidants, N-Acetylcysteine and Elamipretide (SS-31), on Ozone-Induced Airway Hyperresponsiveness and Mucus Hypersecretion

Abstract

Ozone (O₃) exposure induces acute airway injury characterized by airway hyperresponsiveness (AHR) and airway mucus hypersecretion (AMH). Oxidative stress and mitochondria-derived reactive oxygen species (mtROS) are key contributors. We investigated and compared the protective mechanisms of N-acetylcysteine (NAC) and the mitochondria-targeted antioxidant Elamipretide (SS-31) in O₃-induced airway inflammation, AHR and AMH. Wild-type C57BL/6J mice received intraperitoneal NAC or SS-31 1 h before a single O₃ exposure. AHR, bronchoalveolar lavage (BAL) inflammatory cells, mucus production and mucin expression, inflammatory mediators, oxidative stress indices, and PI3K/AKT and NLRP3/caspase-1/GSDMD pathway activation were assessed in vivo. BEAS-2B cells were pretreated with NAC, SS-31, or the PI3K/AKT inhibitor LY294002 before O₃ exposure, and pathway activation was evaluate d in vitro. NAC and SS-31 comparably attenuated O₃-induced AHR, reduced BAL inflammatory cell influx, and decreased AMH and MUC5B expression. Both treatments improved redox balance by reducing ROS/mtROS, lowering malondialdehyde (MDA), increasing superoxide dismutase (SOD) activity, and improving GSH/GSSG. NAC and SS-31 also suppressed O₃-induced inflammatory gene expression and inhibited activation of PI3K/AKT and NLRP3/caspase-1/GSDMD signaling in mouse lungs and BEAS-2B cells. PI3K inhibition recapitulated these protective effects in vitro, supporting a mechanistic role for PI3K/AKT signaling during acute O₃ exposure. NAC and SS-31 protect against acute O₃-induced AHR and AMH by alleviating oxidative stress and suppressing PI3K/AKT-driven inflammatory and pyroptotic pathways. Targeting oxidative stress, including mitochondrial ROS, may represent a viable strategy to mitigate airway damage caused by acute O₃ exposure.