Molecular hydrogen (H₂) dissolved in saline under high pressure (0.6 MPa) to a supersaturated level for 2 hours[¹⁸]. Hydrogen saline was freshly prepared every week. The hydrogen concentration was detected by gas chromatography, the concentration of hydrogen being 0.6 mM in each experiment. Hydrogen saline degassed by gentle stirring was used for control animals.

Male BALB/c mice (20-25 g) were housed in a controlled environment and provided with standard rodent chow and water. To test inhibitory effects on acute inflammation in an animal model, paw edema was induced by subcutaneous injection of 0.05 mL of carrageenan (1%) into the right hind paw, as was previously described [¹⁹]. Right after the carrageenan administration, the animals received an i.p. injection of either saline(5 ml/kg) or hydrogen saline(5 ml/kg). The paw volume was measured by a volume measuring instrument(YLS-7B from Gene&I) at each hour point after edema induction. The increase in percentage of paw volume was calculated based on the volume difference between the normal and abnormal paws (with or without carrageenan injection). Seven animals per group were tested. The Animal and Ethics Review Committee at the Second Military Medical University evaluated and approved the protocol used in this study.

After 4 h of carrageenan treatment, mice were sacrificed, and the tissue of the paws was removed and fixed in alfac solution. Each sample was embedded in paraffin wax, sectioned at 5 μm and stained with hematoxylin-eosin. A representative area was selected for qualitative light microscopic analysis of the inflammatory cellular response with a 20× objective [²⁰]. Five slices from each animal were analyzed and a minimum of three animals for each treatment was taken.

3-nitrotyrosine(3-NT) is the metabolite of the ONOO^-, and it is stable and easier to be detected than ONOO^-. What's more, it can reflect the concentration of ONOO^-. After 4 h of carrageenan treatment, mice were sacrificed. The blood was collected and centrifuged at 3000 rpm. 3-NT concentration was determined in the cell free supernatant by enzyme-linked immunosorbent assay(ELISA). Antibody-matched pairs and respective standards were purchased from R&D system(Minneapolis, Minn, U.S.A), and were used according to the manufacturer's instructions. The sensitivity for the 3-NT ELISA assay is 2 nmol/L.

The activity of tissue MPO was assessed 4 h after injection of carrageenan into the mouse's right hind paw. Samples were placed in 0.75 ml of 80 mM phosphate-buffered saline (PBS), pH 5.4, containing 0.5% hexadecyltrimethylammonium bromide, and were then homogenized (45 s at 0°C) in a motor-driven homogenizer. The homogenate was decanted into a microfuge tube, and the vessel was washed with a second 0.75 ml aliquot of hexadecyltrimethylammonium bromide in buffer. The wash was added to the tube, and the 1.5 ml sample was centrifuged at 12,000 × g at 4°C for 15 min. Triplicate 30 μl samples of the resulting supernatant were added to 96-well microtitre plates. For assay, 200 μl of a mixture containing 100 μl of 80 mM PBS, pH 5.4, 85 μl of 0.22 M PBS, pH 5.4, and 15 μl of 0.017% hydrogen peroxide were added to the wells. The reaction was started with the addition of 20 μl 18.4 mM tetramethylbenzidine HCl in dimethylformamide. Plates were incubated at 37°C for 3 min, and then the reaction was stopped by the addition of 30 μl of 1.46 M sodium acetate. pH 3.0. Enzyme activity was determined colorimetrically using a plate reader (ELX800, BioTech Instruments, Inc.) set to measure absorbance at 630 nm and expressed as mOD/mg tissue.

Macrophages were prepared from BALB/c mice as was previously described [²¹]. Briefly, peritoneal macrophages were harvested from 2 to 3 BALB/c mice, which had been injected intraperitoneally with 1 ml of thioglycollate three days before sterile peritoneal lavage with 5 ml of Hank's balanced salt solution. The collected cells were seeded and cultured in RPMI1640 containing 10% heat-inactivated FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin at a density of 5 × 10⁶ cells/well. The cells were allowed to adhere for 1 h to a 48-well culture plate at 37°C in a 5% CO₂ incubator. Then the cultures were washed twice with RPMI1640 to remove nonadherent cells prior to the addition of 400 μl of fresh RPMI1640 containing 10% FBS. The purity of the adherent macrophages was assessed by Giemsa staining(> 95%).

Total RNA was isolated from macrophages using TriZol Reagent. Total RNA was reverse-transcribed into cDNA and then PCR amplification of the cDNA was performed. The forward and reverse primer sequences of PCR primers were as follows: murine TNF-α: F 5'-CCC TCA CAC TCA GAT CAT CTT CT-3', R 5'-GCT ACG ACG TGG GCT ACA G-3'; murineβ-actin: F 5'-CTT TGC AGC TCC TTC GTT GC-3', R 5'-ACG ATG GAG GGG AAT ACA GC-3' [²²]. An aliquot of the total RNA was reverse-transcribed by RAV2 reverse transcriptase (20 U/ul, TAKARA, Japan) and the oligo-dT primer(300 pmol) in a total volume of 40 ul reaction according to the manufacturer's instructions. cDNAs were analyzed immediately or stored at -20°C until use. Real-time PCR assay was carried out with LightCycler (Roche Diagnostics.Germany) using LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics). 1 ul of cDNA was added to a 19 ul of reaction mixture including MgCl₂ at the optimal concentration, 20 pmol of each primer and 2 ul of a LightCycler FastStart DNA Master SYBR Green I. Then the mixture was incubated in LightCycler under the following conditions: at 95°C for 10 min for denaturation, followed by 50 cycles of 94°C for 1 second, each of the optimal annealing temperature for 5 seconds, 72°C for 10 seconds and finally cooling to 40°C[²³]. We use the double stranded DNA-binding dye SYBR Green I to estimate the expression level of cytokine mRNA[²⁴]. Transcripts of beta-actin, as a housekeeping gene, were quantified as endogenous RNA of reference to normalize each sample. Relative quantities were estimated by the delta-delta-Ct method. The results were normalized as relative expression in which the average value of the TNF-α mRNA was divided by the average value of beta-actin mRNA.

RAW 264.7 murine macrophages were obtained from the American Type Culture Collection (Manassas, USA). These cells and murine peritoneal macrophages were cultured in a 48-well culture plate with 400 μl RPMI1640 in each well and maintained at sub-confluence in a 95% air and 5% CO₂ humidified atmosphere at 37°C [²⁵]. The medium used as the routine subculture was RPMI1640 supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (100 μg/mL). TNF-α concentration was determined in the cell free supernatant obtained after 4 h macrophages co-culture with LPS(100 ng/ml) by enzyme-linked immunosorbent assay(ELISA). Antibody-matched pairs and respective standards were purchased from R&D system(Minneapolis, Minn, U.S.A), and were used according to the manufacturer's instructions. The sensitivity for the TNF ELISA assay is 7 pg/ml.

Statistical comparisons among the groups were assessed by one-way analysis of variance (ANOVA). When F ratios were significant (P < 0.05), LSD 's post hoc tests between two groups were done using SPSS Statistics (SPSS Inc.). P < 0.05 was considered statistically significant.

In order to determine the anti-inflammation activity of hydrogen saline in acute-phase inflammation in vivo, a carrageenan-induced paw oedema experiment was conducted. Hydrogen saline showed a remarkable inhibitory activity in carrageenan-induced paw oedema after carrageenan injection. The time-dependent curve shows that the paw swelling ratio will rise till 4 hours after carrageenan injection. (Figure 1A) Hydrogen saline showed bell shaped effect of down-regulating carrageenan-induced paw swelling, compared with the vehicle control group, which received an equal volume of vehicle only (saline). (Figure 1A.B). And 5 ml/Kg was the most effective dose, which was similar to the results that our lab had observed(5 ml/Kg was the proper dose for the effects)[¹⁸]. It has been reported that the bell shaped phenomenon also exists in the hydrogen gas's effect of anti-ischemia-reperfusion injury[¹²]. This bell shaped phenomenon is quite hard to explain now and its mechanism needs to be studied in our further experiments.

Figure 2 shows some representative photos from the histological changes in the paw tissues following the subplantar injection of carrageenan (0.05 mL/paw) and the anti-inflammation effects of hydrogen saline. The 4 h peak of the carrageenan inflammatory response was found to be associated with subcutaneous oedema along with the heavy infiltration of neutrophils at the site of injection. The treatment of animals with hydrogen saline exhibited a significant decrease in the number of cellular infiltrates.

ONOO^-is unstable and can be deoxidized very soon. 3-NT is stable and easy to be detected, and it can reflect the concentration of ONOO^-. Figure 3 and Table 1 show that the hydrogen saline can decrease the concentration of 3-NT in the plasma.

The neutrophil migration into carrageenan-stimulated mouse paws was indirectly determined by means of MPO activity. Treatment with different doses of hydrogen saline significantly prevented the increase in MPO activity induced by carrageenan (Figure 4, Table 1).

Expression of activated macrophage mRNA was determined 1 hr after the treatment of hydrogen saline or saline. TNF-α mRNA of activated macrophages treated with hydrogen saline was lower than that of those treated with saline.(Figure 5)

The production of TNF-αfrom activated macrophages which were treated with saline proved much more than that of those treated with hydrogen saline. This result was similar in both murine peritoneal macrophages(Figure 6A) and RAW 264.7 murine macrophages (Figure 6B) indicating that hydrogen saline was able to inhibit the TNF-α production of macrophages.

Each value represents the mean ± S.E.M. n = 7. *p < 0.05 **P < 0.01 compared with CA + saline group. Student's t-test after analysis of variance. CA = carrageenan treatment. HS = carrageenan + hydrogen saline treatment.