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The role of Prdx6 in the protection of cells of the crystalline lens from oxidative stress induced by UV exposure.

ABSTRACT: The immediate aim of this study was to investigate alterations in peroxiredoxin (Prdx) 6 at posttranslational levels, and the levels of protein oxidation, lipid peroxidation, and reactive oxygen species (ROS) in lens epithelial cells (LECs) after exposure to severe oxidative stress, such as ultraviolet-B (UV-B). Our ultimate aim was to provide new information on antioxidant defenses in the lens and their regulation, thereby broadening existing knowledge of the role of Prdx6 in lens physiology and pathophysiology.The expression of the hyperoxidized form of Prdx6 and oxidation of protein were analyzed by western blotting and the OxyBlot assay in human LECs (hLECs). ROS levels were quantified using DCFH-DA dye, and cell viability was quantified by the MTS and TUNEL assays. To evaluate the protective effect of Prdx6, we cultured lenses with or without the TAT transduction domain (TAT-HA-Prdx6) and observed (and photographed) the cultures at specified time-points after the exposure to UV-B for the development of opacity.Prdx6 in hLECs was hyperoxidized after exposure to high amounts of UV-B. UV-B treatment of hLECs increased the levels of cell death, protein oxidation, and ROS. hLECs exposed to UV-B showed higher levels of ROS, which could be reduced by the application of extrinsic TAT-HA-Prdx6, attenuating UV-B-induced lens opacity and apoptotic cell death.Excessive oxidative stress induces the hyperoxidation of Prdx6 and may reduce the ability of Prdx6 to protect LECs against ROS or stresses. Because extrinsic Prdx6 could attenuate UV-B-induced abuse, this molecule may have a potential in preventing cataractogenesis.

SUBMITTER: Shibata S

PROVIDER: S-EPMC5544922 | biostudies-other | 2016 Sep

REPOSITORIES: biostudies-other

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Project description:More than 140 million people live and works (in a chronic or intermittent form) above 2500 m worldwide and 35 million live in the Andean Mountains. Furthermore, in Chile, it is estimated that 55,000 persons work in high altitude shifts, where stays at lowlands and interspersed with working stays at highlands. Acute exposure to high altitude has been shown to induce oxidative stress in healthy human lowlanders, due to an increase in free radical formation and a decrease in antioxidant capacity. However, in animal models, intermittent hypoxia (IH) induce preconditioning, like responses and cardioprotection. Here, we aimed to describe in a rat model the responses on cardiac and vascular function to 4 cycles of intermittent hypobaric hypoxia (IHH). Twelve adult Wistar rats were randomly divided into two equal groups, a four-cycle of IHH, and a normobaric hypoxic control. Intermittent hypoxia was induced in a hypobaric chamber in four continuous cycles (1 cycle = 4 days hypoxia + 4 days normoxia), reaching a barometric pressure equivalent to 4600 m of altitude (428 Torr). At the end of the first and fourth cycle, cardiac structural, and functional variables were determined by echocardiography. Thereafter, ex vivo vascular function and biomechanical properties were determined in femoral arteries by wire myography. We further measured cardiac oxidative stress biomarkers (4-Hydroxy-nonenal, HNE; nytrotirosine, NT), reactive oxygen species (ROS) sources (NADPH and mitochondrial), and antioxidant enzymes activity (catalase, CAT; glutathione peroxidase, GPx, and superoxide dismutase, SOD). Our results show a higher ejection and shortening fraction of the left ventricle function by the end of the 4th cycle. Further, femoral vessels showed an improvement of vasodilator capacity and diminished stiffening. Cardiac tissue presented a higher expression of antioxidant enzymes and mitochondrial ROS formation in IHH, as compared with normobaric hypoxic controls. IHH exposure determines a preconditioning effect on the heart and femoral artery, both at structural and functional levels, associated with the induction of antioxidant defence mechanisms. However, mitochondrial ROS generation was increased in cardiac tissue. These findings suggest that initial states of IHH are beneficial for cardiovascular function and protection.

Project description:Thioredoxin (Trx) is an important protein that controls oxidative damage in almost all eukaryotic cells. Trx interaction protein (Txnip) has been reported to negatively regulate the bioavailability of Trx and inhibit its biological function. The E3 ubiquitin ligase ITCH can specifically degrade Txnip via ubiquitination. The apoptosis of human lens epithelial cells (HLECs), which are highly sensitive to redox caused by oxidative stress, is a significant factor for the development of sugar cataract in a high‑glucose environment. However, whether Trx, Txnip and ITCH contribute to the progression of sugar cataracts and the underlying mechanisms remain unknown, and thus, identifying these were the aims of the present study. The present results suggested that the expression levels of Trx, Txnip and ITCH in HLECs cultured with different glucose concentrations were detected by reverse transcription‑quantitative PCR and western blotting, and the apoptotic rate of the cells was detected by flow cytometry and superoxide detection assay. The interaction between ITCH and Txnip was determined by co‑localization immunofluorescence and co‑immunoprecipitation. In addition, a vector and small interfering RNA of ITCH were transfected to overexpress and knockdown ITCH, respectively, to alter the expression of downstream proteins and cell apoptosis. It was found that Txnip was highly expressed in cultured HLECs in high‑glucose environment, and the antioxidative function of Trx was restricted and suppressed, thus promoting apoptosis. The overexpression of ITCH increased the expression of Trx and decreased oxidative stress and apoptosis by decreasing Txnip in cultured HLECs, while downregulation of ITCH significantly decreased the expression of Trx and enhanced oxidative stress and apoptosis. Therefore, the present results indicated that ITCH could regulate the apoptosis of HLECs that were cultured in high‑glucose concentration and that it may be a treatment target for sugar cataract.

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The role of Prdx6 in the protection of cells of the crystalline lens from oxidative stress induced by UV exposure.

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