Background
We have reported that molecular hydrogen (H
2) has potential as a novel antioxidant in preventive and therapeutic applications [
1]. Furthermore, H
2exhibits 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
2in 2007, more than 80 articles involved in H
2have been published to establish the apparent activity of H
2from various medical aspects [
7‐
9].
H
2reacted 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
2by drinking water with dissolved H
2, a small amount of H
2was extensively effective [
10‐
12]; however, it may be difficult to explain that direct reduction of
•OH by a very small amount of H
2reveals all the functions of H
2, because the saturated level of H
2is 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
2was shown to be effective [
14,
15]. Although we have recently shown that H
2can be accumulated with hepatic glycogen, it is unlikely that the amount of H
2is sufficient to exhibit all of its functions [
15].
Moreover, H
2regulated various gene expressions; however, there is no evidence that H
2directly 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
2remains unknown.
ONOO
-is produced by the rapid reaction of nitric monoxide (NO
•)with superoxide anion radicals (
•O
2-) [
18,
19]. We have shown that H
2reduces ONOO
-as well as
•OH [
1]. Different from
•OH, ONOO
-has 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
2functions is caused by reducing cellular ONOO
-.
Here, to verify this hypothesis, we examined protective and regulatory effects of H2on NO•-derived oxidative stress to chondrocytes. We found that H2protected 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 H2functions is responsible for the reduction of ONOO-.
Methods
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 slices were randomly divided into two experimental groups and incubated at 37°C in Dulbecco's modified Eagle's medium (DMEM)/Ham F-12 mixed medium (Gibco Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml).
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
2in the medium by bubbling H
2gas to the saturated level. We also dissolved O
2in a second medium by bubbling O
2gas, and CO
2in a third medium by bubbling CO
2gas. 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
2instead of H
2. The H
2concentration 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, L-penicillamine (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 HistoMark 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.
Table 1
Primers and probes for RT-PCR.
aggrecan | F primer | 5'-GACCAGGAGCAATGTGAGGAG-3' |
| R primer | 5'-CTCGCGGTCGGGAAAGT-3' |
| probe | 5'-CCAAGTTCCAGGGCCACTGTTATCGC-3' |
type II collagen | F primer | 5'-TTGGAGAGACCATGAACGGC-3' |
| R primer | 5'-TTAGCGGTGTTGGGAGCC-3' |
| probe | 5'-CACTTCAGCTACGGCGACGGCAA-3' |
MMP3 | F primer | 5'-TCCCAGGAAAATAGCTGAGAACTT-3' |
| R primer | 5'-AAACCCAAATGCTTCAAAGACAG-3' |
| probe | 5'-CCAGGCATTGGCACAAAGGTGGA-3' |
MMP13 | F primer | 5'-TGGAGTTATGATGATGCTAACCAGAC-3' |
| R primer | 5'-TGTCGCCAATTCCAGGGA-3' |
| probe | 5'-TGGACAAAGACTATCCCCGCCTCATAGAAG-3' |
GAPDH | F primer | 5'-CATCACTGCCACCCAGAAGA-3' |
| R primer | 5'-ATGTTCTGGGCAGCC-3' |
| probe | 5'-TGGATGGCCCCTCTGGAAAGCTG-3' |
Immunoblot analysis
Specimens were homogenized with a micro-homogenizer in SDS (sodium dodecyl sulfate) buffer (1% SDS in PBS), and then centrifuged at 10,000 g for 10 min at 4°C to remove debris. Supernatants were subjected to SDS-PAGE (SDS-polyacrylamide gel electrophoresis) followed by electrotransfer onto a PVDF membrane. The blotted membranes were blocked with Block Ace (DS Pharma Biomedical Co., Ltd.) and incubated with anti-aggrecan polyclonal antibody (Abcam, Cambridge, UK; 1:1,000 dilution), anti-MMP13 polyclonal antibody (Santa Cruz Biotechnology, Inc. 1:1,000 dilution) or anti-actin monoclonal antibody (Sigma-Aldrich; 1:500 dilution) overnight at 4°C. Each band was visualized with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) and an ECL plus Western blotting detection system (GE Healthcare, Piscataway, NJ, USA).
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.
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 ONOO
-formation has been observed in cartilage and subchondral bone in rodent models [
34‐
36] and in cartilage in OA and RA patients [
37‐
39]. ONOO
-induces 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
2regarding ONOO
-in this study.
In this study, we show that H
2protected chondrocytes from death induced by SNAP. SNAP is a donor of NO•; however, NO• has no strong toxicity itself and H
2has no potential to reduce NO•. Our previous study demonstrated that H
2reduces ONOO
-in a cell-free system [
1]. Thus, we speculate that H
2would protect SNAP-treated chondrocytes by decreasing ONOO
-. More importantly, it has been reported that drinking hydrogen water suppress the nitration of kidney proteins, although H
2received from hydrogen water remained for only short period in the organ (less than 5 min) [
11]. In this study, we have shown that H
2in medium suppress the nitration of the chondrocyte proteins (Figure
3). Thus, it is possible that even a very small amount of H
2exhibits anti-oxidative effects by reducing ONOO
-in many situations.
Several laboratories including ours have reported that H
2altered gene expressions involved in inflammation or energy metabolism when animals drank hydrogen water [
15,
17]; however, it is an open question why H
2alters gene expressions, because there is no evidence that H
2directly influences gene expressions. On the other hand, ONOO
-has 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
2consumed by drinking hydrogen water influences nitration in
in vivoexperiments and results in regulatory as well as anti-oxidative effects [
11]. These results agree with the present finding that H
2suppressed the nitration of proteins.
Taken together, this study implies that one of the H2functions, including transcriptional alterations, is caused through reducing ONOO-derived from NO•.
Novel pharmacological strategies aimed at selective removal of ONOO
-may represent a powerful method for preventive and therapeutic use of H
2for joint diseases. Cartilage has no blood vessels and nutrients are supplied through fluid. Since H
2has 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.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SO and ST conceived the experiments. SO and NK designed the actual experiments. TH, TY and NK performed the experiments and data analysis. NK and SO interpreted the data and wrote the paper. All authors have been involved in drafting the manuscript it critically for important intellectual content; and have given final approval of the version to be published.