Possible Role of NRF2 in Cell Response to OZOILE (Stable Ozonides) in Children Affected by Lichen Sclerosus of Foreskin

Abstract

Lichen sclerosus (LS) is a chronic inflammatory disease of the skin, and the gold standard for treatment is the use of the very potent topical steroids, but they can have side effects. Previously, we demonstrated that OZOILE (stable ozonides) were effective in children affected by LS, reducing the inflammatory process and stimulating tissue regeneration of the foreskin, showing a similar efficacy to steroid treatment. In this study, the modulation of inflammatory and oxidative stress pathways was evaluated by qRT-PCR and Western blotting in foreskins affected by LS removed from patients untreated or treated with OZOILE or corticosteroid cream formulations for 7 days before circumcision. OZOILE induced a significant increase in NRF2 and SOD2 levels, while it did not produce change in MIF, NF-kB subunits, and MMPs in comparison to untreated foreskins. Conversely, steroid topical treatment produced a significant reduction in the expression of p65, MIF, and MMP9, but it did not cause variation in NRF2 and SOD2 levels. These results demonstrate that the use of OZOILE as cream formulation exhibits effects on NRF2 signaling, and it does not induce NF-κB activation, unlike corticosteroids. On the basis of our biochemical data, further studies evaluating the role of NRF2 signaling cascade are necessary.

Keywords: OZOILE; inflammation; lichen sclerosus; nuclear factor erythroid 2–related factor 2 (NRF2); ozonated oil.

Figure 1

Evaluation of mRNA levels of NRF2 (A) and NF-kB subunits (B) in foreskin of LS patients untreated (n = 10) or topically treated with either OZOILE (n = 10) or steroids (0.1% mometasone furoate; n = 10). Real-time PCR results (means ± SD) are expressed as relative fold changes in comparison with untreated patients. *** p < 0.001 significant differences in comparison with untreated patients; ^§§§ p < 0.001 significant differences in comparison with steroid-treated patients.

Figure 2

Evaluation of nuclear protein levels of p65 and NRF2 in foreskin of LS patients untreated or topically treated with either OZOILE or steroids (0.1% mometasone furoate). Western blotting results are representative of all tissues analyzed (n = 10 per group), and the densitometric analysis data are expressed as the means ± SD of the samples analyzed (n = 10 per group). *** p < 0.001 significant differences in comparison with untreated patients; ^§§§ p < 0.001 significant differences in comparison with steroid-treated patients.

Figure 3

Evaluation of MIF mRNA levels in the foreskin of LS patients untreated or topically treated with either OZOILE or steroids (0.1% mometasone furoate). Real-time PCR results (means ± SD) are expressed as relative fold changes in comparison with untreated patients. ** p < 0.01 significant differences in comparison with untreated patients; ^§§§ p < 0.001 significant differences in comparison with steroid-treated patients.

Figure 4

Evaluation of protein levels of MMP9 and SOD2 in the foreskin of LS patients untreated or topically treated with either OZOILE or steroids (0.1% mometasone furoate). Real-time PCR results (means ± SD) are expressed as relative fold changes in comparison with untreated patients (A,B). The protein amounts were assessed by Western blot analysis (C). Western blotting is representative of all tissues analyzed (n = 10 for each group), and densitometric analysis data represent the means ± SD of analyzed samples (n = 10 for each group). * p < 0.05 and *** p < 0.001 significant differences in comparison with tissues from untreated patients; ^§ p < 0.05 and ^§§§ p < 0.001 significant differences in comparison with steroid-treated patients.

Figure 5

NF-κB and NRF2 pathways activation. The dotted lines highlights the possible link between NF-κB and NRF2 pathways in response to a stimuli.