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

We treated cultured chondrocytes from porcine hindlimb cartilage or from rat meniscus fibrecartilage with a donor of NO？, S-nitroso-N-acetylpenicillamine (SNAP) in the presence or absence of H2. 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.SNAP treatment increased the levels of nitrated proteins. H2 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. H2 restoratively increased the gene expressions of aggrecan and type II collagen in the presence of H2. Conversely, the gene expressions of MMP3 and MMP13 were restoratively down-regulated with H2. Thus, H2 acted to restore transcriptional alterations induced by ONOO-.These results imply that one of the functions of H2 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 H2 for joint diseases.We have reported that molecular hydrogen (H2) has potential as a novel antioxidant in preventive and therapeutic applications [1]. Furthermore, H2 exhibits not only anti-oxidative stress effects [2,3], but also has various anti-inflammatory [4,5] and anti-allergic effects [6]. Since the publication of the first article on the biological contribution of H2 in 2007, more than 80 articles involved in H2 have been published to establish the apparent activity of H2 from various medical aspects [7-9].H2 reacted with strong reactive oxygen/nitrogen species including hydroxyl radical and peroxinitrite (ONOO-) in cell-free reactions and protected c