Recently ozone therapy has been applied successfully as adjuvant therapy in the treatment of several disorders such as rheumatoid arthrıtis, heart and vascular disease, asthma emphysema and multiple sclerosis. Positive effects of ozone therapy have been hypothesized to rely on the capability of mild acute oxidative stress to induce antioxidant response. Nevertheless, molecular mechanisms behind those beneficial effects remain unclear. Some evidence suggests that mild oxidative stress induced by ozone treatments leads to activation of the transcription factor Nrf2 (nuclear factor erythroid-derived-like2). Nrf2 regulates the expression of genes under the control of antioxidant response element (ARE) enhancer. These genes include drug metabolizing enzymes, such as glutathione s-transferases (GSTs) and NAD(P)H-quinone Oxidoreductase 1 (NQO1), and antioxidant genes, such as heme oxygenase-1 (OH-1), the subunit of γ-glutamylcysteine synthetase (γ-GCS) and thioredoxin. Under basal conditions, Nrf2 is sequestered in the cytoplasm by its specific inhibitor Keap1 (Kelch-like ECH associated protein), which promotes Nrf2 ubiquitination and proteasomal degradation within a few minutes after its transcription. Under specific stimuli, Nrf2 dissociates from Keap1 and is preserved from degradation. Then, Nrf2 translocates into the nucleus, heterodimerizes with Maf proteins and trans-activates ARE-mediated genes. The current study was designed to investigate whether ozone therapy might induce ARE activation via Nrf2-dependent mechanisms. First, we established a HeLa cell line engineered to express NanoLuc Luciferase under the control of ARE promoter. We show that the treatment with different concentrations of ozone increased ARE-driven expression of NanoLuc in a dose- 413 dependent manner (Figure 1a). Ultrastructural immunocytochemistry showed Nrf2 to be associated to perichromatin fibrils, which are sites of active transcription (Figure 1b). Hela cells were transiently transfected with Nrf2-GFP and/or Keap1-dsRed fusion proteins to enable microscopy visualization of Nfr2 and Keap1 subcellular localization (Figure 2a). As expected, in untreated cells Nrf2-GFP disappeared when co-transfected with Keap1-dsRed. In contrast, ozone treatment induced persistence of Nrf2-GFP fluorescence and its massive nuclear translocation, suggesting that ozone might prevent Keap1-mediated Nrf2 degradation. In line with this conclusion we found an increased amount of Nrf2 protein compared to controls over 30 minutes after ozone treatment (Figure 2b), likely due to an increased Nrf2 stability. Finally, Keap1 transient overexpression partially reverted ARE-Luc activation in ozone treated cells (Figure 2c), thus providing further evidence that ozone treatment is able to induce an antioxidant stress response through a Keap1/Nrf2-dependent pathway.
|Titolo:||Ozone treatment induces antioxidant stress response through Keap1/Nrf2 dependent pathway|
|Data di pubblicazione:||2017|
|Appare nelle tipologie:||04.01 Contributo in atti di convegno|