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.

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.

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.

Eruca sativa (rocket salad; arugula) is a well-known edible plant commonly used in salads and as a topping for pizza and pasta. In this study, one new cyclic thiocarbamate (1) and four new kaempferol glycosides (2–5), along with 15 known compounds (6–20), were isolated from leaves of E. sativa using chromatographic methods. The structures of all compounds were elucidated based on their HR-ESI-MS, ¹H and ¹³C NMR, and 2D NMR data. All isolates were evaluated for their cardioprotective and antiplatelet activities. Among them, compound 2 exhibited the most potent antioxidant activity in a DPPH assay. Further investigation revealed that compound 2 significantly reduced cellular ROS levels in H9c2 cardiomyocytes under hypoxia/reoxygenation conditions and effectively decreased apoptotic cell death in H9c2 cells, as determined by Annexin V-FITC/PI staining. Additionally, compound 2 demonstrated dose-dependent inhibition of CRP-induced platelet aggregation in human washed platelets, suggesting its potential as a dual-functional candidate for cardiovascular protection.

Doxorubicin (DOX) is a widely used anticancer drug associated with severe cardiotoxicity primarily driven by mitochondrial dysfunction and reactive oxygen species (ROS) production. Hydrogen peroxide (H₂O₂), a major mitochondrial ROS, significantly contributes to cardiotoxicity, yet its precise mechanistic role in DOX-induced cardiotoxicity remains incompletely understood. Here, we investigated the critical role of mitochondrial H₂O₂ in DOX-induced cardiotoxicity using rat cardiomyocyte H9c2 cells with stable knockdown or reconstitution of mitochondrial antioxidant enzyme peroxiredoxin Ⅲ (PrxⅢ), which specifically regulates mitochondrial H₂O₂ levels. Our results demonstrated that severe mitochondrial H₂O₂ accumulation (>10-fold compared to control) in PrxⅢ-depleted cells exacerbated mitochondrial oxidative stress, evidenced by increased cardiolipin oxidation and mitochondrial membrane potential dissipation. Furthermore, excessive mitochondrial H₂O₂ impaired mitochondrial fusion by reducing fusion-related protein expression, disrupted autophagic flux via lysosomal dysfunction, and significantly attenuated mitophagy, ultimately leading to enhanced apoptosis. Conversely, moderate mitochondrial H₂O₂ levels (5- to 8-fold increase compared to control) observed in PrxⅢ-expressing cells promoted mitochondrial elongation, enhanced mitophagy, and preserved autophagic flux, suggesting a protective adaptation against DOX-induced oxidative stress. In vivo experiments using PrxⅢ knockout mice confirmed that loss of PrxⅢ aggravated DOX-induced cardiac dysfunction. Bioinformatic analysis of independent public transcriptome datasets and targeted qPCR validation in PrxⅢ-deficient cardiac tissues further confirmed that the mitochondrial quality control pathways identified in vitro are robustly dysregulated in vivo. Additionally, PrxⅢ deficiency markedly increased mitochondrial structural damage without significantly affecting cardiac fibrosis or hypertrophy. Notably, mitigating the mitochondrial H₂O₂ burden and protecting the mitochondrial inner membrane using the mitochondria-targeted antioxidant peptide SS-31 successfully rescued this exacerbated cardiac dysfunction. In conclusion, our findings establish mitochondrial H₂O₂ as a pivotal determinant in DOX-induced mitochondrial dysfunction and cardiotoxicity, highlighting PrxⅢ as a promising therapeutic target for mitigating oxidative cardiac injury.