Molecular hydrogen protects chondrocytes from oxidative stress and indirectly alters gene expressions through reducing peroxynitrite derived from nitric oxide

Background: Molecular hydrogen (H-2) functions as an extensive protector against oxidative stress, inflammation and allergic reaction in various biological models and clinical tests; however, its essential mechanisms remain unknown. H-2 directly reacts with the strong reactive nitrogen species peroxynitrite (ONOO-) as well as hydroxyl radicals (center dot OH), but not with nitric oxide radical (NO center dot). We hypothesized that one of the H-2 functions is caused by reducing cellular ONOO-, which is generated by the rapid reaction of NO center dot with superoxides (center dot O-2(-)). To verify this hypothesis, we examined whether H-2 could restore cytotoxicity and transcriptional alterations induced by ONOO(-)derived from NO center dot in chondrocytes. Methods: We treated cultured chondrocytes from porcine hindlimb cartilage or from rat meniscus fibrecartilage with a donor of NO center dot, S-nitroso-N-acetylpenicillamine (SNAP) in the presence or absence of H-2. Chondrocyte viability was determined using a LIVE/DEAD Viability/Cytotoxicity Kit. Gene expressions of the matrix proteins of cartilage and the matrix metalloproteinases were analyzed by reverse transcriptase-coupled real-time PCR method. Results: SNAP treatment increased the levels of nitrated proteins. H-2 decreased the levels of the nitrated proteins, and suppressed chondrocyte death. It is known that the matrix proteins of cartilage (including aggrecan and type II collagen) and matrix metalloproteinases (such as MMP3 and MMP13) are down- and up-regulated by ONOO-, respectively. H-2 restoratively increased the gene expressions of aggrecan and type II collagen in the presence of H-2. Conversely, the gene expressions of MMP3 and MMP13 were restoratively down- regulated with H-2. Thus, H-2 acted to restore transcriptional alterations induced by ONOO-. Conclusions: These results imply that one of the functions of H-2 exhibits cytoprotective effects and transcriptional alterations through reducing ONOO-. Moreover, novel pharmacological strategies aimed at selective removal of ONOO- may represent a powerful method for preventive and therapeutic use of H-2 for joint diseases.