Nonsteroidal anti-inflammatory drug (NSAID) use is associated with adverse consequences, including hepatic injury. The detrimental hepatotoxicity of diclofenac, a widely used NSAID, is primarily connected to oxidative damage in mitochondria, which are the primary source of reactive oxygen species (ROS). The primary ROS responsible for inducing diclofenac-related hepatocellular toxicity and the principal antioxidant that mitigates these ROS remain unknown. Peroxiredoxin III (PrxIII) is the most abundant and potent H₂O₂-eliminating enzyme in the mitochondria of mammalian cells. Here, we investigated the role of mitochondrial H₂O₂ and the protective function of PrxIII in diclofenac-induced mitochondrial dysfunction and apoptosis in hepatocytes. Mitochondrial H₂O₂ levels were differentiated from other types of ROS using a fluorescent H₂O₂ indicator. Upon diclofenac treatment, PrxIII-knockdown HepG2 human hepatoma cells showed higher levels of mitochondrial H₂O₂ than PrxIII-expressing controls. PrxIII-depleted cells exhibited higher mitochondrial dysfunction as measured by a lower oxygen consumption rate, loss of mitochondrial membrane potential, cardiolipin oxidation, and caspase activation, and were more sensitive to apoptosis. Ectopic expression of mitochondrially targeted catalase in PrxIII-knockdown HepG2 cells or in primary hepatocytes derived from PrxIII-knockout mice suppressed the diclofenac-induced accumulation of mitochondrial H₂O₂ and decreased apoptosis. Thus, we demonstrated that mitochondrial H₂O₂ is a key mediator of diclofenac-induced hepatocellular damage driven by mitochondrial dysfunction and apoptosis. We showed that PrxIII loss results in the critical accumulation of mitochondrial H₂O₂ and increases the harmful effects of diclofenac. PrxIII or other antioxidants targeting mitochondrial H₂O₂ could be explored as potential therapeutic agents to protect against the hepatotoxicity associated with NSAID use.

Aims: NADPH oxidase (NOX)-derived reactive oxygen species (ROS) are critical for platelet activation and thrombus formation. We hypothesized that inhibiting NOX-mediated ROS production with a pan-NOX inhibitor, APX-115, could effectively suppress platelet activation and thrombus formation, potentially serving as a novel antiplatelet therapeutic. This study aimed to explore the effects of APX-115 on human platelet functional responses and ROS-mediated signaling pathways. Results: APX-115 inhibited intracellular and extracellular ROS production in collagen-stimulated platelets, suppressing aggregation, P-selectin exposure, and ATP release. By preserving protein tyrosine phosphatase activity, APX-115 reduced tyrosine phosphorylation-dependent pathways inhibition, including spleen tyrosine kinase, LAT, Vav1, Bruton’s tyrosine kinase, and phospholipase Cγ2, leading to decreased PKC activation and calcium mobilization. APX-115 also suppressed collagen-induced integrin αIIbβ3 activation, accompanied by elevated cGMP and vasodilator-stimulated phosphoprotein phosphorylation levels. In addition, APX-115 reduced p38 MAPK and ERK5 activation, leading to diminished phospholipase A2 phosphorylation, thromboxane production, and the exposure of procoagulant phosphatidylserine. These inhibitory effects extended to thrombus development caused by platelet adherence under shear and arterial thrombosis without prolonging bleeding time in murine models. Innovation: This study is the first to demonstrate that APX-115 inhibits NOX-mediated ROS production, platelet activation, and thrombus formation. By uncovering its effects on collagen receptor glycoprotein VI-mediated pathways, the work highlights the promise of APX-115 as an antiplatelet and antithrombotic agent. Conclusion: Our findings highlight the therapeutic potential of APX-115 in treating thrombotic and cardiovascular disorders by targeting NOX-mediated ROS production to mitigate platelet hyperreactivity and thrombus formation. Antioxid. Redox Signal. 43, 288–307.

Peroxiredoxin Ⅲ (PrxⅢ) is a mitochondria-localized peroxidase that plays a key role in detoxifying hydrogen peroxide (H₂O₂) and preserving organelle homeostasis. While its antioxidant function is well established under physiological conditions, the role of PrxⅢ in the context of cardiac hypoxia/reoxygenation (H/R) injury remains incompletely understood. In this study, we investigated the protective function of PrxⅢ in cardiomyocytes exposed to H/R stress, a widely used in vitro model to mimic ischemia/reperfusion injury. Using H9c2 cells and primary neonatal rat cardiomyocytes, we found that PrxⅢ knockdown significantly increased mitochondrial H₂O₂ accumulation, leading to excessive mitochondrial fragmentation, impaired mitophagy, and reduced cell survival following H/R. Western blot analysis revealed that mitophagy regulators Parkin and BNIP3 were upregulated under moderate oxidative stress but were markedly suppressed in PrxⅢ-deficient cells after H/R, indicating that mitophagy activation is sensitive to the degree of oxidative stress. These findings were confirmed in vivo using mt-Keima transgenic mice, which showed significantly reduced mitophagic flux in PrxⅢ knockout hearts subjected to ischemia/reperfusion. In addition, PrxⅢ loss impaired lysosomal acidification and proteolytic activity, further contributing to defective autophagic flux. Re-expression of PrxⅢ restored mitochondrial morphology, mitophagy activity, and lysosome function, highlighting its central role in maintaining mitochondrial quality control (MQC). Collectively, our results demonstrate that PrxⅢ mitigates mitochondrial oxidative damage and preserves MQC by coordinating mitochondrial dynamics, mitophagy, and lysosomal integrity. These findings suggest that PrxⅢ may serve as a promising therapeutic target for preventing cardiac injury induced by oxidative stress during ischemia/reperfusion.

Mefenamic acid (MFA) is a commonly prescribed non-steroidal anti-inflammatory drug (NSAID) with anti-inflammatory and analgesic properties. MFA is known to have potent antioxidant properties and a neuroprotective effect against oxidative stress. However, its impact on the liver is unclear. This study aimed to elucidate the antioxidative effects of MFA and their underlying mechanisms. We observed that MFA treatment upregulated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Treatment with various anthranilic acid derivative-class NSAIDs, including MFA, increased the expression of sequestosome 1 (SQSTM1) in HepG2 cells. MFA disrupted the interaction between Kelch-like ECH-associated protein 1 (Keap1) and Nrf2, activating the Nrf2 signaling pathway. SQTM1 knockdown experiments revealed that the effect of MFA on the Nrf2 pathway was masked in the absence of SQSTM1. To assess the cytoprotective effect of MFA, we employed tert-Butyl hydroperoxide (tBHP) as a ROS inducer. Notably, MFA exhibited a protective effect against tBHP-induced cytotoxicity in HepG2 cells. This cytoprotective effect was abolished when SQSTM1 was knocked down, suggesting the involvement of SQSTM1 in mediating the protective effect of MFA against tBHP-induced toxicity. In conclusion, this study demonstrated that MFA exhibits cytoprotective effects by upregulating SQSTM1 and activating the Nrf2 pathway. These findings improve our understanding of the pharmacological actions of MFA and highlight its potential as a therapeutic agent for oxidative stress-related conditions.

Ultraviolet B (UVB) irradiation generates reactive oxygen species (ROS), which can damage exposed skin cells. Mitochondria and NADPH oxidase are the two principal producers of ROS in UVB-irradiated keratinocytes. Peroxiredoxin V (PrxV) is a mitochondrial and cytosolic cysteine-dependent peroxidase enzyme that robustly removes H₂O₂. We investigated PrxV’s role in protecting epidermal keratinocytes against UVB-induced ROS damage. We separated mitochondrial and cytosolic H₂O₂ levels from other types of ROS using fluorescent H₂O₂ indicators. Upon UVB irradiation, PrxV-knockdown HaCaT human keratinocytes showed higher levels of mitochondrial and cytosolic H₂O₂ than PrxV-expressing controls. PrxV depletion enhanced hyperoxidation-mediated inactivation of mitochondrial PrxIII and cytosolic PrxI and PrxII in UVB-irradiated keratinocytes. PrxV-depleted keratinocytes exhibited mitochondrial dysfunction and were more susceptible to apoptosis through decreased oxygen consumption rate, loss of mitochondrial membrane potential, cardiolipin oxidation, cytochrome C release, and caspase activation. Our findings show that PrxV serves to protect epidermal keratinocytes from UVB-induced damage such as mitochondrial dysfunction and apoptosis, not only by directly removing mitochondrial and cytosolic H₂O₂ but also by indirectly improving the catalytic activity of mitochondrial PrxIII and cytosolic PrxI and PrxII. It is possible that strengthening PrxV defenses could aid in preventing UVB-induced skin damage.