Rotenone, a classical inhibitor of mitochondrial complex I, disrupts electron transport and promotes the generation of reactive oxygen species (ROS), contributing to inflammation and cell death. However, the precise molecular mechanisms linking mitochondrial dysfunction to inflammatory signaling remain incompletely understood. In this study, we investigated the role of the cGAS–STING pathway in rotenone-induced NLRP3 inflammasome activation in PMA-differentiated THP-1 macrophages. Rotenone treatment activated the cGAS–STING axis, as evidenced by increased cGAS expression and the phosphorylation of STING and TBK1. This activation led to the nuclear translocation of NF-κB and the upregulation of NLRP3, promoting inflammasome priming and IL-1β secretion. Inhibition of STING using H-151 markedly suppressed NLRP3 expression, NF-κB activation, and IL-1β release. Similarly, cyclosporin A, an inhibitor of mitochondrial permeability transition pore opening, reduced mitochondrial ROS, cytosolic oxidized mitochondrial DNA, and downstream activation of the cGAS–STING pathway, thereby attenuating inflammasome activation. These findings demonstrate that rotenone activates the NLRP3 inflammasome via mitochondrial ROS-mediated release of mtDNA and subsequent activation of the cGAS–STING–NF-κB signaling axis in THP-1-derived macrophages.

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.

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.

The original version of this article unfortunately contained a mistake in the article title.