PMC

Hydrogen decreases nitrotyrosine in chondrocytes and cartilage matrix. (A) Meniscus fibrocartilage from SD rats was incubated with 1 mM SNAP in the presence or absence of hydrogen for 3 hr at 37°C. Frozen sections were stained with anti-nitorotyrosine antibody and visualized with DAB as described in Methods. The inset shows chondrocytes. Cartilage incubated with 1 mM SIN-1 and without SNAP was used for positive and negative staining controls, respectively. Scale bar: 40 μm. (B) Levels of nitrotyrosine in cartilage were estimated from anti-nitrotyrosine immunostaining using an image analysis program, Image J program. Data are the mean ± SD (n = 4). *p < 0.05; control versus hydrogen in 1 mM SNAP-treated groups.

Hydrogen alters mRNA and protein expressions of matrix proteins and matrix-metalloproteases (MMPs). Meniscus fibrocartilage from rats was incubated with 1 mM SNAP in the presence or absence of hydrogen for 4 hr or 20 hr at 37°C. Total RNA was extracted from 4 hr-incubated cartilage and the expression levels of aggrecan (A), type II collagen (B), GAPDH (C), MMP3 (D) and MMP13 (E) were analyzed by real-time PCR coupled with reverse transcription. Data are the mean ± SD (n = 4). *p < 0.05; **p < 0.01. (F) Total protein was extracted from 20 hr-incubated cartilage and the expression levels of aggrecan, MMP13 and actin were analyzed by immunoblotting.

Hydrogen protects chondrocytes of hyaline cartilage from cell death. (A) Porcine cartilage slices were incubated with 0, 1 or 3 mM SNAP in the presence or absence of hydrogen for 12 hr at 37°C. Cells were stained with a mixture of calcein AM (Live cell: green) and ethidium homodimer (Dead cell: red) as described in Materials and methods. Scale bar: 100 μm. (B) Chondrocyte viability was determined by counting green and red cells from three areas of each slice. Six slices were used for each experimental group. The slices were incubated with 0, 1 or 3 mM SNAP in the presence or absence of hydrogen for 12, 24 or 36 hr at 37°C. Data are the means ± SD (n = 6). *p < 0.05; **p < 0.01.

Hydrogen protects chondrocytes of fibrocartilages from cell death. (A) Meniscus fibrocartilages from SD rats was incubated with 1 mM SNAP in the presence or absence of hydrogen for 0, 6, 20, 48, or 80 hr at 37°C. Cells were stained with calcein AM (Live cell: green) and ethidium homodimer (Dead cell: red) as described in Materials and methods. Scale bar: 40 μm. (B) Chondrocyte viability was determined by counting green and red cells from three regions of each slice. Six slices were used for each experimental group. The slices were incubated with 1 mM SNAP in the presence or absence of hydrogen for the indicated periods at 37°C. Data are the means ± SD (n = 6). *p < 0.05; ***p < 0.001. (C) The slices were incubated with 0, 0.3, 1, or 3 mM SNAP in the presence or absence of hydrogen for 48 hr at 37°C. Data are the means ± SD (n = 6). *p < 0.05; ***p < 0.001.