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Effect of Aqueous Ozone on the NF-B System

¹ Department of Restorative Dentistry & Periodontology,
³ Department of Orthodontics,
⁴ Experimental Surgery & Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University, Goethe Street 70, 80336 Munich, Germany;
² Institute of Clinical Chemistry & Pathobiochemistry, Klinikum rechts der Isar, Technische Universität München, Germany; and
⁵ Institute of Clinical Chemistry, Medizinische Hochschule Hannover, Germany

View larger version (24K): [in this window] [in a new window]   Figure 1. Activation of NF-B was inhibited in the presence of O3 medium. (A) BHY cells were pre-incubated with serum-free O3 medium (15 min; the ozonation state is indicated in %) before TNF was added (20 ng/mL, 45 min), and an electrophoretic mobility shift assay was performed (NF-B and Sp-1 binding). The asterisk marks a control reaction in which a 100-fold concentration of unlabeled consensus oligonucleotide was added. (B) Cells were treated with O3 medium (HGF-1, 45 min; HeLa, 1 hr), followed by stimulation with TNF (HGF-1, 5 ng/mL for 45 min; HeLa, 1 ng/mL for 15 min). NF-B activity normalized to Sp-1 is shown (densitometric analysis). NF-B activity after TNF stimulation following pre-incubation with non-ozonized medium was defined as 100% (dashed line) (n = 3, mean ± SD). (C) Periodontal ligament tissue separated into equal-sized portions was incubated (1 hr, 37°C) with serum-free non-ozonized medium (indicated by "–") or O3 medium (100% ozonation state, indicated by "+"), followed by two-hour incubation in fresh medium without ozone. The samples were shock-frozen and homogenized, and nuclear extracts were prepared. Left: Electrophoretic mobility shift assays were performed (NF-B and Sp-1 binding). The asterisk marks a control reaction in which a 100-fold concentration of unlabeled consensus oligonucleotide was added. Right: Densitometrically measured NF-B activity was normalized to Sp-1 (n = 3, mean ± SD).

View larger version (25K): [in this window] [in a new window]   Figure 2. Effect of O3 medium on IB proteolysis, NF-B-dependent gene expression, and B-dependent transcription. (A) IB proteolysis was prevented in O3-medium-cultivated cells. BHY (left panel) and HeLa cells (right panel) were pre-incubated with serum-free O3 medium (BHY, 15 min; HeLa, 1 hr) (ozonation state in %) before TNF was added (BHY, 20 ng/mL, 45 min; HeLa, 1 ng/mL, 15 min). IB and actin were determined by Western blot analysis. (B) and (C) NF-B target gene expression was inhibited by pre-incubation with O3 medium. (B) BHY and HGF-1 cells were pre-incubated with serum-free O3 medium (BHY, 15 min; HGF-1, 45 min), followed by TNF (BHY, 20 ng/mL, 45 min; HGF-1, 5 ng/mL, 45 min). The supernatant was then replaced by regular medium, and interleukin-8 was measured after 5 hrs by an enzyme-linked immunosorbent assay (BHY, n = 2; HGF-1, n = 3; mean ± SD). (C) BHY cells were pre-treated with O3 medium (15 min). The supernatant was replaced by regular medium, TNF (20 ng/mL, 45 min) was added, and interleukin-1β was measured after 12 hrs (immunoassay). The interleukin-1β level after TNF stimulation following pre-treatment with non-ozonized medium was defined as 100% (dashed line) (n = 3, mean ± SD). (D) B-dependent transcription was inhibited by pre-incubation with O3 medium. HeLa cells were transfected with pGL2-3B-Luc and the Renilla plasmid. After 24 hrs, cells were treated with serum-free O3 medium (1 hr), followed by TNF (1 ng/mL, 15 min). The supernatant was replaced by regular medium, and relative luciferase activity was measured after 5 hrs (n = 3, mean ± SD).

View larger version (41K): [in this window] [in a new window]   Figure 3. O3 amino acids selectively inhibited the NF-B system. (A) HeLa cells were incubated with O3-phosphate-buffered saline (1 hr), followed by TNF stimulation (1 ng/mL, 15 min). The ozonation state is indicated in %. We performed an electrophoretic mobility shift assay to determine NF-B activity as well as Sp-1 binding. (B) Cells were pre-incubated (1 hr) with non-ozonized or ozonized amino acids (medium concentration) dissolved in phosphate-buffered saline (100% ozonation state), followed by stimulation with TNF (1 ng/mL, 15 min). The activation of NF-B was determined by an electrophoretic mobility shift assay. (C) Densitometric analysis of the NF-B activity normalized to Sp-1 is shown (n = 3, mean ± SD). NF-B activity of cells pre-incubated with non-ozonized amino acids in phosphate-buffered saline and stimulated with TNF was defined as 100% (dashed line). (D) HeLa cells were pre-incubated with non-ozonized or ozonized glucose (medium concentration), followed by TNF stimulation. The NF-B activity compared with Sp-1 binding was determined by an electrophoretic mobility shift assay.

View larger version (23K): [in this window] [in a new window]   Figure 4. Inhibition of NF-B by aqueous ozone. In this study, pre-incubation with aqueous ozone (O3 medium) led, in TNF-stimulated cells, to an inhibition of IB proteolysis (Western blot analysis), NF-B DNA binding (electrophoretic mobility shift assay), and B-dependent transcription (luciferase assay), as well as to reduced expression of the target genes interleukin-8 and -1β (immunoassay). This indicates that aqueous ozone inhibited NF-B signaling at the level and/or upstream of IB. In the Fig., the classic NF-B dimer is depicted, consisting of the subunits p65 and p50. The activation pathway of NF-B is described in more detail in the INTRODUCTION.

Journal of Dental Research, Vol. 86, No. 5, 451-456 (2007)
DOI: 10.1177/154405910708600512


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