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

Background Molecular hydrogen (H2) 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. H2 directly reacts with the strong reactive nitrogen species peroxynitrite (ONOO-) as well as hydroxyl radicals (•OH), but not with nitric oxide radical (NO•). We hypothesized that one of the H2 functions is caused by reducing cellular ONOO-, which is generated by the rapid reaction of NO• with superoxides (•O2-). To verify this hypothesis, we examined whether H2 could restore cytotoxicity and transcriptional alterations induced by ONOO- derived from NO• in chondrocytes. Methods 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. Results 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-. Conclusions 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.


Background
We have reported that molecular hydrogen (H 2 ) has potential as a novel antioxidant in preventive and therapeutic applications [1]. Furthermore, H 2 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 H 2 in 2007, more than 80 articles involved in H 2 have been published to establish the apparent activity of H 2 from various medical aspects [7][8][9].
H 2 reacted with strong reactive oxygen/nitrogen species including hydroxyl radical and peroxinitrite (ONOO -) in cell-free reactions and protected cultured cells depending upon the decrease of hydroxyl radicals (•OH) [1]. Subsequent and recent experiments including ours indicated that a small amount of hydrogen is also effective against various stimuli [8,9]. When model animals consumed H 2 by drinking water with dissolved H 2 , a small amount of H 2 was extensively effective [10][11][12]; however, it may be difficult to explain that direct reduction of •OH by a very small amount of H 2 reveals all the functions of H 2 , because the saturated level of H 2 is only 0.8 mM and the dwelling time of •OH is very short in the body [11,13]. In fact, drinking 0.04 or 0.08 mM H 2 was shown to be effective [14,15]. Although we have recently shown that H 2 can be accumulated with hepatic glycogen, it is unlikely that the amount of H 2 is sufficient to exhibit all of its functions [15].
Moreover, H 2 regulated various gene expressions; however, there is no evidence that H 2 directly reacts with factors involved in transcriptional regulation including FGF21 [15], inflammatory cytokines [11], HMGB1 [16], and HO-1 [17]. It remains unclear whether such regulations are the cause or consequence of the effects against oxidative stress. Moreover, the primary molecular target of H 2 remains unknown.
ONOOis produced by the rapid reaction of nitric monoxide (NO•) with superoxide anion radicals (•O 2 -) [18,19]. We have shown that H 2 reduces ONOOas well as •OH [1]. Different from •OH, ONOOhas a longer lifespan and the potential to regulate gene expression through nitration of target proteins [20,21]. Thus, we hypothesized that one of the H 2 functions is caused by reducing cellular ONOO -.
Here, to verify this hypothesis, we examined protective and regulatory effects of H 2 on NO•-derived oxidative stress to chondrocytes. We found that H 2 protected chondrocytes from oxidative stress, and alternated gene expressions, contrary to the manner of transcriptional regulation by ONOO -. This study implies that at least one of the H 2 functions is responsible for the reduction of ONOO -.

Cartilage slice culture
A fresh hindlimb of a slaughtered male seven-month-old pig was purchased from Tokyo Shibaura Organ Co., Ltd. (Minato-ku, Tokyo, Japan). There were no possible contaminant diseases. Cartilage from the healthy porcine hindlimb (metatarsophalangeal joint) was cut into pieces for culture (2 mm width × 7 mm length × full thickness) as described previously [22]. Male Sprague-Dawley rats of 10 weeks of age were purchased from Nippon SLC (Hamamatsu, Shizuoka, Japan). Cartilage from the meniscus of a rat was also sliced into pieces (full width × full length × 0.5 mm thickness) for culture. Since the meniscus structure is not uniform and the peripheral part contains fewer chondrocytes, we used slices prepared from the middle part of the meniscus.
The care and use of laboratory animals were in accordance with the NIH guidelines. This study was approved by the Animal Care and Use Committee of Nippon Medical School (Bunkyo-ku, Tokyo, Japan).

Hydrogen treatment
We prepared H 2 -dissolved culture medium as described previously [1]. In brief, we dissolved H 2 in the medium by bubbling H 2 gas to the saturated level. We also dissolved O 2 in a second medium by bubbling O 2 gas, and CO 2 in a third medium by bubbling CO 2 gas. We combined these media to give a medium consisting of 75% H 2 , 20% O 2 , 5% CO 2 (vol/vol/vol). We then cultured the cartilage slices in a closed culture flask filled with the medium. Control medium contained 75% N 2 instead of H 2 . The H 2 concentration was maintained for 24 hr as described [15].

Cell death assay
The cartilage slices were incubated for 12 -80 hr in medium containing 0.3 -3 mM S-nitroso-N-acetyl-D, Lpenicillamine (SNAP) (Cayman Chemical, Ann Arbor, MI, USA) in the presence or absence of H 2 [22,23]. Chondrocyte viability was determined using a LIVE/ DEAD Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR, USA). Living, dying and dead cells were stained with green, yellow (combination of green and red) and red fluorescence, respectively, and visualized with a confocal scanning laser microscope (FLUOVIEW FV300; Olympus, Tokyo, Japan).

Immunohistochemical staining
Frozen sections of 6 μm-thick were fixed with 10% formalin and treated with 0.3% hydrogen peroxide in methanol to inhibit endogenous peroxidase activity. The sections were incubated with 10% Block Ace (DS Pharma Biomedical Co., Ltd., Suita, Osaka, Japan) in phosphate buffered saline (PBS) and then incubated with anti-nitrotyrosine monoclonal antibody (Calbiochem, San Diego, CA, USA; 1:100 dilution with 10% Block Ace in PBS) overnight at 4°C. Nitrotyrosine residues were visualized with DAB using horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) and a Histo-Mark ORANGE kit (KPL, Gaithersburg, MD, USA). As a positive control for staining, we used sections from cartilage treated with 1 mM 3-morpholinosydnonimine (SIN-1) (Sigma-Aldrich, St. Louis, MO, USA), which generates both superoxide anion and nitric oxide that spontaneously produce peroxynitrite. The positive area was estimated using the Image J program (version 1.41; National Institutes of Health, Bethesda, MD, USA) from four sections for each group.

RNA isolation and RT-PCR
Total RNA was isolated from the cartilage using an RNeasy Mini kit (QIAGEN, Valencia, CA, USA). Complementary DNA synthesized by SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) was analyzed by quantitative PCR using the Thermal Cycler Dice Real Time System TP800 (TAKARA BIO Inc., Otsu, Shiga, Japan). All samples were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Primer and probe sequences for each PCR are listed in Table 1.

Statistical analysis
We performed statistical analysis using StatView software (SAS Institute) by applying an unpaired two-tailed Student's t-test and ANOVA followed by Fisher's exact test, as described previously [1]. Differences were considered significant at p < 0.05.

H 2 protects chondrocytes of hyaline and fibrecartilage from cell death
It is reported that cultured chondrocytes are sensitive to exposure to SNAP, a donor of NO• [22] and that H 2 exhibits no direct reaction with NO• in cultured cells as well as in a cell-free reaction. To verify the hypothesis that H 2 protects cells by reducing ONOO -, we examined the effect of H 2 on cell death induced by SNAP by using hyaline cartilage slices from a porcine hindlimb metatarsophalangeal joint as a target. Chondrocyte viability was determined using a LIVE/DEAD Viability/Cytotoxicity Kit, which provides quantitative analyses of the proportion of live and dead cells in a mixed population ( Figure 1A). In living cells, membrane-permeated calcein AM is cleaved by esterases to yield cytoplasmic green fluorescence, and in dead cells membrane-impermeable ethidium homodimer-1 labels nucleic acids with red fluorescence. Dying cells, whose membrane structure has been disrupted but still have some esterase activity, were double stained as yellow. We counted green, red and yellow cells for statistical analysis (Additional file 1: Table S1). Cell viability was calculated as the percentage of green cell numbers against total cell numbers ( Figure 1B). Significant protection of chondrocytes by H 2 was observed in the treatment with 3 mM SNAP for 12 hr ( Figure 1A and 1B). More evident effects were obtained with longer SNAP treatment ( Figure 1B).
Next, we examined another type of cartilage, meniscus fibrecartilage, isolated from rats instead of swine specimens. Because it is easier to isolate swine than rat cartilage, we used swine cartilage for preliminary experiments; however, for further analysis, cartilage from rats is more suitable for RNA and protein analysis because genomic databases and antibodies are available. Treatment with 1 mM SNAP induced cell death in a time-dependent manner and H 2 suppressed chondrocyte death at each time point (Figure 2A and 2B, Additional file 2: Table S2). H 2 significantly protected chondrocytes from death with various concentrations of SNAP treatment for 48 hr ( Figure  2C, Additional file 3: Table S3). These results indicate that H 2 protects chondrocytes by stimuli derived from NO•, although H 2 has no potential to react with NO•.

H 2 decreases nitrotyrosine in chondrocytes and matrix of cartilage induced by SNAP
ONOOis a strong modifier of nitration in proteins. To confirm that H 2 decreased ONOOderived from NO•, Table 1 Primers and probes for RT-PCR. we examined levels of nitrotyrosine residues in cartilage immunohistologically. In fact, NO• increased the levels of nitrotyrosine, and H 2 restored its increase ( Figure 3). Thus, H 2 should decrease ONOOderived from NO•.

H 2 restores down-regulation of matrix expression and upregulation of matrix-metallo protease expression induced by SNAP
It was also reported that ONOOdown-regulates gene expressions of the cartilage matrix proteins including aggrecan and type II collagen [24]. Conversely, levels of matrix-metallo protease are known to be up-regulated by ONOO - [24]. We then investigated the effect of H 2 on the expression of chondrocyte-specific matrix genes.
Isolated meniscus fibrecartilage was incubated in DMEM/F-12 supplemented with 10% FBS with or without 1 mM SNAP in the presence or absence of H 2 . The levels of mRNA for the matrix proteins of type II collagen and aggrecan core protein were quantified with real-time PCR coupled with reverse transcription ( Figure  4A and 4B). Indeed, SNAP down-regulated aggrecan and collagen II gene expressions as expected. The decreased gene expressions of the matrix proteins were significantly restored by H 2 -dissolved culture medium, suggesting that the decreased ONOOrestored the gene expression. The possibility cannot be ruled out that oxidative damage derived from SNAP may reduce any gene expressions in a non-specific manner. We examined therefore the effect of H 2 on catabolic enzyme genes induced by SNAP, because levels of matrix-metallo protease are known to be contradictorily up-regulated by ONOO - [24]. The levels of MMP-3 and MMP-13 mRNA were measured with quantitative real-time PCR after treatment with SNAP with or without H 2 ( Figure 4D and 4E). Moreover, the alterations of the aggrecan and MMP-13 proteins corresponded to their mRNA levels ( Figure 4F). Thus, SNAP up-regulated MMP-3 and MMP-13 gene expressions as expected, whereas H 2 significantly suppressed MMP gene expressions, suggesting that H 2 restored the increased expressions by decreasing ONOO -.

Discussion
Joint diseases including osteoarthritis (OA) and rheumatoid arthritis (RA) are the most common disabling diseases, especially among elderly people. Arthritis is a degenerative disease involving abnormalities in chondrocytes, articular cartilage and other joint tissue, and is mediated by a number of underlying biochemical and physical stimuli [25,26]. Recent studies revealed that oxidative stress plays a leading role in the initiation and progression of the disease process [27,28]. As a joint disease model of aged patients, we stimulated chondrocytes with oxidative stress derived from NO•. The cartilage consists mostly of the extracellular matrix, which is synthesized by chondrocytes [28,29]. The extracellular matrix is composed of collagens and proteoglycans that are responsible for the important compressive and tensile properties of cartilage [28]. The major oxidative stress generated by chondrocytes is one of the most powerful oxidants ONOO -, which was produced by the rapid reaction of NO• with •O 2 - [18,19]. At an earlier stage, NO• has been considered as the primary inducer of chondrocyte death [30]; however, it has been revealed that the oxidative strength of NO• is not sufficient to initiate cell death [31,32]. A series of experiments have indicated that the major cytotoxicity attributed to NO• is rather due to ONOO - [20,33]. Increased ONOOformation has been observed in cartilage and subchondral bone in rodent models [34][35][36] and in cartilage in OA and RA patients [37][38][39]. ONOOinduces cell death and regulates the decreased expression of collagens and proteoglycans and increased matrix metallo proteinases in chondrocytes, resulting in matrix degradation [24,40]. Thus, chondrocyte is a suitable target for investing the effect of H 2 regarding ONOOin this study.
In this study, we show that H 2 protected chondrocytes from death induced by SNAP. SNAP is a donor of NO•; however, NO• has no strong toxicity itself and H 2 has no potential to reduce NO•. Our previous study demonstrated that H 2 reduces ONOOin a cell-free system [1]. Thus, we speculate that H 2 would protect SNAPtreated chondrocytes by decreasing ONOO -. More importantly, it has been reported that drinking hydrogen water suppress the nitration of kidney proteins, although H 2 received from hydrogen water remained for only short period in the organ (less than 5 min) [11]. In this study, we have shown that H 2 in medium suppress the nitration of the chondrocyte proteins ( Figure 3). Thus, it is possible that even a very small amount of H 2 exhibits anti-oxidative effects by reducing ONOOin many situations.
Several laboratories including ours have reported that H 2 altered gene expressions involved in inflammation or energy metabolism when animals drank hydrogen water [15,17]; however, it is an open question why H 2 alters gene expressions, because there is no evidence that H 2 directly influences gene expressions. On the other hand, ONOOhas the potential to regulate gene expressions through the nitration of factors involved in transcriptional regulation [20]. As mentioned above, drinking hydrogen water suppresses the nitration of proteins; thus, it is possible that the very small amount of H 2 consumed by drinking hydrogen water influences nitration in in vivo experiments and results in regulatory as well as anti-oxidative effects [11]. These results agree with the present finding that H 2 suppressed the nitration of proteins.
Taken together, this study implies that one of the H 2 functions, including transcriptional alterations, is caused through reducing ONOOderived from NO•.
Novel pharmacological strategies aimed at selective removal of ONOOmay represent a powerful method for preventive and therapeutic use of H 2 for joint diseases. Cartilage has no blood vessels and nutrients are supplied through fluid. Since H 2 has a great advantage to rapidly diffuse into tissues even without blood flow [41,42], it may be useful to prevent joint diseases by reducing oxidative stress and by suppressing the decrease in matrix proteins and inhibiting degradation by proteinases.

Conclusions
This study implies that one of the H 2 functions, including transcriptional alterations, is caused through reducing ONOOderived from NO•. Novel pharmacological strategies aimed at selective removal of ONOOmay represent a powerful method for preventive and therapeutic use of H 2 for joint diseases.