Date Published: March 27, 2017
Publisher: Public Library of Science
Author(s): Yanwei Li, Haifeng Liu, Wei Zeng, Jing Wei, Hemachandra Reddy.
An increase in the osmolarity of tears induced by excessive evaporation of the aqueous tear phase is a major pathological mechanism behind dry eye. Exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction, and apoptosis. Edaravone, a hydroxyl radical scavenging agent, is clinically used to reduce neuronal damage following ischemic stroke. In this study, we found that treatment with hyperosmotic media at 400 and 450 mOsM increased the levels of ROS and mitochondrial oxidative damage, which were ameliorated by edaravone treatment in a dose-dependent manner. We also found that edaravone could improve mitochondrial function in HCEpiCs by increasing the levels of ATP and mitochondrial membrane potential. MTT and LDH assays indicated that edaravone could attenuate hyperosmolarity-induced cell death. It was found that edaravone prevented apoptosis by decreasing the level of cleaved caspase-3, and attenuating the release of cytochrome C. Mechanistically, we found that edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1, and GCLC.
As a multifactorial disease of the tears and ocular surface, dry eye affects more than 20% of the adult population worldwide. This condition is characterized by inadequacy of the tear film that protects the eyes, thereby having an adverse effect on eye health and comfort . An increase in tear osmolarity induced by increased evaporation of the aqueous tear phase has been considered as a major pathological mechanism behind dry eye. Previous in vivo and in vitro studies have shown that hyperosmolarity initiates inflammation of the ocular surface and cellular apoptosis in dry eye patients, dry eye mouse models, and in in vitro hyperosmotic culture models of human corneal epithelial cells (HCECs) . Notably, increased levels of ROS, lipid oxidative stress markers, and inflammatory cells have been found in the conjunctiva and tear film of Sjögren’s syndrome patients, suggesting that oxidative stress plays a critical role in the pathogenesis of dry eye [3, 4]. In addition, hyperosmolarity has been recognized as a pro-inflammatory stress factor for the corneal epithelium. Hyperosmolarity leads to an increase in the activation of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK) and p38, which are serine/threonine-specific protein kinases that respond to extracellular stimuli and regulate various cellular activities in the corneal epithelium [5, 6]. Importantly, exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to apoptosis . Preventing hyperosmolarity-mediated toxicity has become an important therapeutic strategy for the treatment of dry eye.
Edaravone inhibited the production of ROS stimulated by hyperosmolarity in HCEpiCs as evaluated via DCFH-DA assay. The results indicate that fluorescence intensity was markedly increased after cells were treated with hyperosmotic media at 400 and 450 mOsM for 24 h (Fig 1), while prophylactic treatment with 10 μM or 20 μM edaravone attenuated the stimulated ROS production in a dose-dependent manner.
Tear hyperosmolarity plays a central role in ocular surface damage related to dry eye , and oxidative stress is an important contributing factor to hyperosmolarity. A recent study showed that hyperosmolarity induces oxidative stress in HCECs by stimulating the generation of ROS and suppressing protein levels of the antioxidant enzymes SODl and GPXl, which in turn causes a significant increase in cellular oxidative damage biomarkers resulting from lipid peroxidation of cell membranes (HNE and MDA) and in mitochondrial DNA (8-OHdG and aconitase-2). In addition, both in vivo and in vitro studies have shown that hyperosmotic stress induces the expression and production of proinflammatory cytokines in cells on the ocular surface . Notably, hyperosmolarity-induced ocular surface cell death is a key mitochondria-mediated event in inflammatory eye diseases. Hyperosmolarity has been reported to induce mitochondrial depolarization and stimulate mitochondrial cell death in human corneal epithelial (HCE-T) cells . Countless types of natural and synthesized chemical agents have been tested for the prevention of hyperosmolar stress on corneal epithelial cells. The key finding of the current study is that edaravone, a powerful free radical scavenger, inhibited hyperosmolarity-induced oxidative stress, mitochondrial dysfunction, and apoptosis.
In the present work, we demonstrated the protective action of edaravone in hyperosmotic culture models of human corneal epithelial cells (HCECs). The drug partly reversed alterations in the generation of ROS and mitochondrial oxidative stress induced by hyperosmolarity, and also improved mitochondrial dysfunction. In addition, edaravone treatment attenuated hyperosmolarity-induced cell death and apoptosis. Mechanically, edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1 and GCLC. These findings strongly suggest that edaravone could be a potential candidate for the treatment of hyperosmolarity-induced eye diseases.