Molecular hydrogen regulates gene expression by modifying the free radical chain reaction-dependent generation of oxidized phospholipid mediators

We previously showed that H2 acts as a novel antioxidant to protect cells against oxidative stress. Subsequently, numerous studies have indicated the potential applications of H2 in therapeutic and preventive medicine. Moreover, H2 regulates various signal transduction pathways and the expression of many genes. However, the primary targets of H2 in the signal transduction pathways are unknown. Here, we attempted to determine how H2 regulates gene expression. In a pure chemical system, H2 gas (approximately 1%, v/v) suppressed the autoxidation of linoleic acid that proceeds by a free radical chain reaction, and pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC), one of the major phospholipids, was autoxidized in the presence or absence of H2. H2 modified the chemical production of the autoxidized phospholipid species in the cell-free system. Exposure of cultured cells to the H2-dependently autoxidized phospholipid species reduced Ca2+ signal transduction and mediated the expression of various genes as revealed by comprehensive microarray analysis. In the cultured cells, H2 suppressed free radical chain reaction-dependent peroxidation and recovered the increased cellular Ca2+, resulting in the regulation of Ca2+-dependent gene expression. Thus, H2 might regulate gene expression via the Ca2+ signal transduction pathway by modifying the free radical-dependent generation of oxidized phospholipid mediators.

We assumed that low concentrations of H 2 would influence some chemical reactions leading to the production of putative oxidized lipid mediators for the modulation of signal transduction. Because PAPC is one of the major phospholipids in mammalian biomembranes, the role of H 2 in the chemical production of oxidized phospholipid mediators was determined by conducting autoxidation of pure PAPC (resulting in OxPAPC) in the absence of any catalysts in the dark. The peroxidation of PAPC in air was confirmed by an increase in the signal for the fluorescent dye specific to lipid peroxides, Liperfluo (Fig. 3a). A previous study indicated that OxPAPC activates transcription factors involved in Ca 2+ signaling 16 . Indeed, when THP-1 cells (a human monocytic cell line derived from a patient with acute monocytic leukemia) were exposed to OxPAPC, a transient increase in cellular Ca 2+ was observed when a Ca 2+ -sensitive fluorescent dye, Fluo4-AM was used (Fig. 3b). This Ca 2+ signaling depended on OxPAPC in an oxidation time-dependent manner (Fig. 3c). Next, the H 2 -dependent production of OxPAPC, which leads to the activation of Ca 2+ signaling, was investigated by autoxidizing PAPC for 3 days at 25 °C in air at various concentrations of H 2 (designated as H 2 OxPAPC, and the notation of H 2 [x%]OxPAPC was used when autoxidized in the presence of x% H 2 ). H 2 suppressed the generation of total peroxides as revealed by Liperfluro fluorescence intensity (Fig. 3d). Ca 2+ signaling was observed when PAPC was autoxidized with less than 0.3% H 2 , whereas more than 1.3% H 2 significantly disrupted this signaling (Fig. 3e).
In order to investigate the molecule(s) influenced by H 2 , we analyzed H 2 OxPAPCs by using mass spectrometry on autoxidation day 3. In all, 209 bands were detected, with molecular masses ranging from 126.3754 to 991.6494 Da; this was consistent with the findings of a previous report 15 (Supplementary Fig. 1). The differences in the production of H 2 OxPAPC and OxPAPC species were presented using a heat map ( Supplementary Fig. 1i). The levels of many bands were increased or decreased with differences in concentrations of H 2 . For examples as the relatively increased species, the levels of the Ca 2+ signaling inducers POVPC 16 , HOOA-PC, HOdiA-PC, and hydroxyeicosatetraenoic acid-3-phosphocholine (HETE-PC) 17 were slightly increased in response to H 2 ( Supplementary Fig. 1i).
Because the reduced form of POVPC was reported to function as an antagonist 18 , it is possible that increased levels of the reduced form(s) of some OxPAPC species, rather than the decreased levels of putative agonists (such as POVPC), might have disrupted Ca 2+ signaling as a putative antagonist(s). Further studies are warranted to identify the H 2 -dependent bioactive mediator(s).
Comprehensive analysis of H 2 -dependent regulation of gene expression. Next, we investigated how H 2 OxPAPC influences gene expression. PAPC was autoxidized in the absence or presence of various concentrations of H 2 for 3 days and then administered to cultured THP-1 cells. In a preliminary experiment, the change in the expression level of tumor necrosis factor (TNF)-α gene in response to OxPAPC from that to H 2 OxPAPC peaked at 4 h. Thus, by using microarray analysis, we comprehensively analyzed the change in gene expression in response to the H 2 -dependent mediators at 4 h in three samples under each condition. In all, 86 genes were selected according to the following criteria as described in the legend of Fig. 4a Table 1). The gene expression profile was presented in a heat map (Fig. 4a). The selected genes were validated by semi-quantitative real-time polymerase chain reaction (RT-PCR), and marginal changes in the expression levels of some genes were confirmed ( Supplementary Fig. 2).
In addition, the regulatory expression of TNF-α and IL-8 by H 2 OxPAPC was investigated using THP-1 and a different cell type (human aortic endothelial cells: HAEC), respectively (Fig. 4b,c).
According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database (http://www. genome.jp/kegg/pathway.html), the functions of 7,143 genes were identified and classified (Fig. 4d, upper). We classified the 86 selected genes (Fig. 4e, lower). Of these 86 genes, 46.5% belonged to those involved in signaling pathways (Fig. 4d, upper), whereas 25.8% of the total number of 7,143 genes is involved in signaling pathways (Fig. 4d, lower). Genes encoding factors involved in signal transduction and transcription factors are indicated by blue and black, respectively, on the right in the heat map (Fig. 4a).
Among the genes involved in signaling pathways, the proportion of those belonging to Ca 2+ signaling were lower in the selected genes than in those in the entire genome, indicating that H 2 regulates fewer components of the Ca 2+ signaling pathways (Fig. 4e, lower). This was consistent with the finding that H 2 OxPAPC decreased Ca 2+ signaling. In contrast, the proportion of genes belonging to the mitogen-activate protein kinase (MAPK)  signaling was higher (Fig. 4e, lower), indicating that H 2 regulates more components of MAPK signal transduction pathways (Fig. 4e, lower). The signal transduction pathways that were regulated by H 2 are shown in Supplementary Table 1 according to the KEGG Pathway Database. These data suggested the possibility that low concentrations of H 2 contribute to various signal transduction pathways via oxidized phospholipid species.
cAMP response element binding protein (CREB)-target genes were selected according to the CREB Target Gene Database (http://natural.salk.edu/CREB/), and nuclear factor of activated T cells (NFAT) target genes were selected by referring to Medline, as shown in Supplementary Table 1. The target genes of CREB and NFAT are marked by red on the right in the heat map panel as NFAT or CREB (Fig. 4a). A considerable number of the selected genes were targets of CREB or NFAT (Fig. 4 f). These data are consistent with the findings of previous studies showing the Ca 2+ -dependent regulation by these transcription factors: CREB is activated via phosphorylation by a calmodulin-dependent kinase (CaMK) 19 in a Ca 2+ -dependent manner, and NFAT is dephosphorylated by calcineurin (CN) in a Ca 2+ -dependent manner, translocates to the nucleus, and then functions as a transcription factor with its partner proteins, e.g., activator protein 1 (AP-1), CREB, or nuclear factor-kappa B (NF-κ B) 20 . Indeed, exposure of THP-1 to OxPAPC, but not to H 2 OxPAPC, stimulated the nuclear translocation of NFAT ( Supplementary Fig. 3).
Thus, H 2 -dependent oxidized mediators or putative antagonists could be associated with transcriptional regulation via Ca 2+ signaling.
Free radical inducers contributed to the NFAT pathway in cultured cells. Autoxidation of unsaturated fatty acids, including PAPC, proceeds by a free radical chain reaction 13 , and·OH is the primary trigger for this reaction 13,21,22 . We previously showed that H 2 reduces ·OH levels inside cultured cells by using the spin trapping method and a specific fluorescent indicator 1 . Thus, in this study, we investigated the effects of H 2 on the lipid free radical chain reaction by using cultured cells. To initiate a free radical chain reaction inside the cells, we used 2,2′ -azobis(2-methylpropionamidine)dihydrochloride (AAPH) 23 , which is not affected by H 2 ( Supplementary  Fig. 4) and is suitable for the slow generation of free radicals by a spontaneous chemical reaction. The lipid free radical chain reaction results in the production of lipid peroxides (LPOs) 21,24 , which can be detected using the fluorescent dye Liperfluo 25 . Thus, we exposed cultured THP-1 cells to AAPH and estimated LPO production based on the Liperfluo signal. The Liperfluo signal significantly decreased in the presence of low levels of H 2 gas (e.g., 1.3%; Fig. 5a,b). Thus, even at such low concentrations, H 2 has the potential to reduce the generation of LPOs by suppressing the initiation and/or propagation of free radical chain reactions in cultured cells.
Next, we determined whether the responses induced by chemically produced H 2 OxPAPC (Figs 3 and 4) could simulate the effects induced by the free radicals in cultured cells. When THP-1 cells were exposed to AAPH, the cellular Ca 2+ levels increased (Fig. 5c) in a time-dependent manner (Fig. 5d), as shown by the analysis of Fluo-3, and the Ca 2+ signaling was suppressed by H 2 (Fig. 5c,e). NFAT was also activated, as shown by the translocation of NFAT into the nucleus (Fig. 5f,g), and the nuclear translocation of NFAT were recovered by H 2 (Fig. 5f,g). Moreover, the free radical inducer stimulated the expression of some target genes of NFAT, including TNF-α, early growth response protein 1 (EGR1), and activating transcription factor 3 (ATF3), which have been shown in Supplementary Table 1, and H 2 decreased their expressions (Fig. 5h), suggesting that H 2 regulates these genes via the NFAT pathway.
In contrast, AAPH-mediated activation of CREB was not observed ( Supplementary Fig. 5) in this cultured cell line, regardless of the stimulation of cellular Ca 2+ . In particular, the expression of the CREB-target gene NFKB2 (NF-κ B, subunit 2 gene) was not affected by AAPH (Fig. 5i), and the expression of HMOX1 (Heme Oxygenase 1 gene), a nuclear factor-E2-related factor 2 (Nrf2)-target, was slightly but not significantly increased by H 2 (Fig. 5i). This result was consistent with those of a previous study 26 . Thus, the NFAT pathway could mainly contribute to the H 2 -dependent transcriptional response induced by free radicals at least in THP-1 cells.
Taken together, these cellular responses, at least partly, are in agreement with those obtained using the in vitro H 2 -dependent products of OxPAPC species (Figs 3, 4). Therefore, we proposed a model in which H 2 is linked to the modulation of Ca 2+ signal transduction and the NFAT pathway via oxidized phospholipid species, as illustrated in Fig. 6.

Discussion
While the biological effects of H 2 have been evaluated in more than 300 animal studies and 10 clinical analyses in humans 6,7 , the molecular mechanisms by which H 2 at low concentrations exerts its multiple effects on signal transduction remained unknown. Therefore, in this study, we aimed to examine how H 2 regulates signal transduction pathways that mediate gene expression. Our results suggested that low concentrations of H 2 modulated Ca 2+ signal transduction and regulated gene expression by modifying the production of oxidized phospholipid species. Hence, these data provide important insights into one of the molecular mechanisms by which H 2 mediates gene expression. H 2 can be ingested via several methods. Drinking of H 2 -infused water (H 2 -water) has been shown to be efficacious in the treatment of various diseases in animal models and humans 6,7 ; however, H 2 can be infused up to only 0.8 mM under atmospheric pressure, and drinking saturated H 2 -water provides a blood concentration up to only ~10 μ M, with a short dwelling time in the body 11,27 . Moreover, inhaling 1%-4% (v/v) H 2 gas was shown to be effective, reaching concentrations of 8-32 μ M H 2 in the blood 1,4,5 . However, initiation of cellular signals by these low concentrations of H 2 may be difficult to be explained because H 2 should be too inert to react with most molecules. To activate H 2 for reaction with the other molecules, a sufficient level of a putative catalyst must be present; however, it is unlikely that such a putative catalyst would be abundant inside cells. Moreover, H 2 is very small and is unlikely to bind to a putative H 2 -binding receptor because its intra-molecular fluctuation would be expected to lead to instability in terms of thermodynamics, as previously discussed 28 . Thus, it was unknown how low concentrations of H 2 regulate signal transduction and gene expression.
Since increased oxidative stress involving ·OH triggers free radical chain reactions, we assumed that the chemically produced mediators derived from phospholipids could contribute to various pathogenic conditions. In the present study, we verified that a small amount of H 2 (as low as 1.3%) affected free radical-dependent lipid peroxidation, from which oxidized lipid mediators should be derived 22 .
Generally, H 2 hydrogenates unsaturated fatty acids at higher temperatures with a palladium catalyst. To the best of our knowledge, no studies have examined autoxidation-dependent hydrogenation at approximately 1% (v/v) H 2 gas at 37 °C without any catalysts. Although H 2 was thought to be inert in the absence of a catalyst at body temperature, we demonstrated that approximately 1% (v/v) H 2 suppressed autoxidation of an unsaturated fatty acid in a chemically pure system in this study; thus, our data provided insights into the biological activities of H 2 .
There are two possibilities: the effects of oxidized phospholipid species on Ca 2+ signaling may be explain by decreased levels of a putative agonist that induces Ca 2+ signaling or by increased levels of a putative antagonist that disturbs Ca 2+ signaling. Although we could not identify these species in this study, it is likely that H 2 modified the production of reduced forms of oxidized phospholipid species during free radical chain reactions by the following previous findings: POVPC is a bioactive phospholipid-mediator that is produced by chemical oxidation of PAPC, and the reduced form of POPVC has been shown to function as an antagonist for signal transduction 18 . Thus, it is possible that during a lipid free radical chain reaction, H 2 contributes to the generation of a reduced form(s) that function(s) as an antagonist(s). Therefore, we proposed a hypothetic model in which H 2 is linked to the modulation of Ca 2+ signal transduction and the NFAT pathway via oxidized phospholipid species as illustrated in Fig. 6.
Previous studies have shown that 1%-4% was efficacious in inhaling H 2 gas in various animal experiments 1,3,4,29-31 . Since a mixed gas containing 1.3% H 2 , 30% O 2 and 68.7% N 2 is available, the effects of around 1.3% needed to be investigated in further studies, including clinical ones 5 . The effective concentrations of H 2 gas were approximately consistent throughout this study .
No receptors involved in Ca 2+ signaling were identified in the present study; however, a previous study showed that some chemically oxidized phospholipid mediators, such as 9-HODE and 11-HETE, could bind a G-protein coupled receptor (G2A) to induce Ca 2+ signaling 17 . Thus, putative oxidized phospholipid mediators or antagonists might bind to G-protein coupled receptors to modulate signal transduction.
In addition to the anti-oxidative roles of H 2 , it has shown to function as an immunosuppressant in allograft transplantation 32 . This immunosuppressant effect can be explained by the suppression of NFAT activation because an immunosuppressant such as CsA and tacrolimus (FK506) acts through the inactivation of calcineurin. Since pro-inflammatory cytokines are regulated by NFAT-dependent mechanisms 20 , the anti-inflammatory effects by H 2 can be explained by the suppression of NFAT. Additionally, the anti-allergic effects of H 2 can be explained by the decrease in Ca 2+ /NFAT signaling 33 .
A considerable number of the multiple functions of H 2 , as shown by previous studies, might be explained by the link between H 2 and NFAT because of the numerous multiple functions of NFAT 20,34 . For example, decreased expression of inducible nitric oxide synthase (iNOS) by H 2 35 can be explained by the inactivation of NFAT 36 . The suppression of osteoclast differentiation 37 and improvement of hypertension 38,39 by H 2 could involve the NFAT pathway 40,41 . Moreover, the decreased expression of gene products through an NFAT-dependent pathway might be involved in α -synuclein-induced degeneration of midbrain dopaminergic neurons in Parkinson's disease 42 . This NFAT-dependent pathway might explain the beneficial effects of H 2 in these patients 8 . Further studies are needed to elucidate the mechanisms by which H 2 exerts multiple functions in terms of the involvement of the NFAT pathway.
In summary, in this study, we investigated the link among H 2 , oxidized phospholipids, and Ca 2+ signaling. Further studies are warranted to identify the H 2 -dependent bioactive mediator(s). Our data provided important insights into one of the mechanisms by which H 2 regulates signal transduction and gene expression; however, H 2 might contribute to other types of signaling pathways as well because H 2 regulates many genes belonging to various signaling pathways. A more detailed understanding of the molecular mechanisms of H 2 -dependent signal transduction and gene expression is expected to facilitate the application of H 2 in a wide range of medical applications.

Methods
Measurement of H 2 . Gases containing H 2 were prepared by mixing H 2 , O 2 , N 2 , and CO 2 at various concentrations from each gas cylinder equipped with a flow meter. The H 2 concentration in the mixed gas or air was tested in each experiment by using gas chromatography (Breath Gas Analyzer, Model TGA2000; TERAMECS Co. Ltd., Kyoto, Japan) as described previously 1 . For the measurement of H 2 in the solvent, H 2 was transferred to the air phase in a closed aluminum bag, and the concentration of H 2 measured by using gas chromatography treated with 10 mM AAPH for 3 h in the presence of the indicated concentrations of H 2 . The translocation of NFAT into the nucleus was examined as described in Methods and shown by immunostaining in yellow. The nucleus was counter-stained with Hoechst 33342 as shown in blue. Scale bar: 50 μ m. (g) The NFAT-expressing areas were semi-quantified and shown by the ratio of NFAT in the nucleus with that in cytosol. *P = 0.023 and **P < 0.01 vs. no H 2 (n = 10). (h,i) The expressions of the NFAT-target genes (TNF-α, EGR1, and ATF3) (h) and non-NFAT target gens (NFKB2 and HMOX1) (i) were estimated using RT-PCR coupled with a TaqMan probe (the probes are listed in Supplementary Table 2). The names of the genes are described in Supplementary Table  1. *P = 0.015 (for ATF3) (+ AAPH and + H 2 vs. + AAPH and -H 2 ). #P = 0.14 (for HMOX1) (+ AAPH and -H 2 vs. + AAPH and + H 2 ), and **P < 0.01 (n = 3) Scientific RepoRts | 6:18971 | DOI: 10.1038/srep18971 as described previously 1 . The aluminum used in the bag was covered with a plastic film to avoid any influence of aluminum.
Autoxidation of linoleic acid-film. Linoleic acid and (± )9-HODE were purchased from Nacalai Tesque (Kyoto, Japan) and CAY (MI, USA), respectively. Linoleic acid was dissolved in cyclohexane to 16 mM, and 2 μ L was dispensed into each glass tube (φ 10 × 50 mm) that had been filled with argon gas; it was allowed to dry up to form a linoleic acid-film at the bottom of a glass tube. The glass tubes were placed into a closed aluminum bag, and the gas in the bag was completely replaced with the indicated mixed gas, where pure H 2 , O 2 , and N 2 were obtained from separate cylinders. The bag was incubated at 37 °C for 20 h for the autoxidation, and 0.2 mL cyclohexane was immediately added to the glass tube to obtain 0.16 mM peroxidized linoleic acid. The concentration of conjugated diene was estimated by measuring the absorption at 234 nm while scanning from 200 to 300 nm.
Autoxidation of pure PAPC in air in the absence or presence of H 2 . Chemically synthesized pure PAPC was purchased from Avanti Polar Lipids (Alabaster, AL, USA). PAPC was autoxidized in air as described previously 43 . Briefly, 0.5 mg of PAPC in 50 μ L of chloroform was transferred to a φ 10 × 50 mm glass tube and dried up under a gentle stream of nitrogen. The lipid residue was allowed to autoxidize in air with 100% humidity at 25 °C in the presence or absence of the indicated concentrations of H 2 gas in a closed aluminum bag for the indicated periods, and then suspended in PBS at a concentration of 0.5 mg/mL.

Estimation of OxPAPC with Liperfluo.
OxPAPC was assayed in ethanol with Liperfluo as described previously 25 . Five min after adding OxPAPC to 1 μ M Liperfluo at room temperature, the fluorescence was measured using a fluorescence spectrophotometer (RF-5300PC; Shimadzu Corporation, Kyoto, Japan), where wavelengths of excitation and emission were set at 488 and 535 nm, respectively.  115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl 2 , 1.8 mM CaCl 2 , 13.8 mM glucose) containing 0.064% pluronic F-127 and 1.25 mM probenecid for 30 min at 37 °C. The cells were washed with PBS and resuspended in recording medium containing 1.25 mM probenecid. The cells were seeded on 35-mm glass-bottomed dishes and then stimulated with 100 μ g/mL OxPAPC or H 2 OxPAPC, followed by 25 μ M ATP. The changes in Fluo 4-AM fluorescence were monitored using a laser scanning confocal microscope (FV1200; Olympus Corporation, Tokyo, Japan). The strength of each fluorescent signal in 400 cells was examined and judged as positive if there was greater than 30% of the ATP signal.

Measurement of Ca
Intracellular Ca 2+ of THP-1 cells treated with the free radical inducer AAPH 23 was measured by Fluo-3 (F-23915; Molecular Probes, Eugene, OR, USA). Briefly, THP-1 cells were pre-incubated with 2 μ M Fluo 3-AM in HBSS containing 0.02% pluronic F-127 for 30 min at 37 °C, resuspended in RPMI1640 (with 10% FBS) containing 2.5 mM probenecid, seeded in 24-well plates, and then treated with AAPH in the presence or absence of H 2 . Changes in Fluo-3 fluorescence signals were observed using a laser scanning confocal microscope (FV1200; Olympus).
Mass spectrometric analysis and presentation of data using heat maps. OxPAPC (dissolved in chloroform at 2.5 mg/mL) was analyzed using by electrospray ionization-mass spectrometry (ESI-MS) by using an LTQ ORBITRAP XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a nitrogen sheath gas flow rate of 40 AU at 300 °C. The sample was directly infused. The scanning range was from m/z 250 to 1000 in the positive ion detection mode. The ion spray voltage was set to 4 kV. OxPAPC species were identified according to their m/z values and confirmed using mass spectrometric analysis as described previously 14,44,45 .
Two independent experiments were performed. The average of the data was used for construction of a heat map and displayed in mass spectrometric profiles. In the heat map, bands were arranged according to molecular mass from small to large, and the strength of each band obtained from H 2 OxPAPC was compared with those by OxPAPC. Red and green bands represented increased and decreased levels as compared with those of OxPAPC, respectively. The mass spectrometric display indicates the average band from two experiments. Only when bands were detected by all of 10 experiments (two experiments at 0%, 0.2%, 0.3%, 1.3% and 5% of H 2 ), they were adopted.
Comprehensive analysis of gene expression. THP-1 cells were exposed for 4 h to PAPC or OxPAPC, H 2 [1.3%]OxPAPC, and H 2 [5%]OxPAPC that had been autoxidized for 3 days with 0%, 1.3%, or 5% H 2 , respectively. Total RNA was extracted using an RNeasy Mini Kit according to the manufacturer's protocol (Qiagen, Valencia, CA, USA) and labeled using a Low-Input QuickAmp Labeling Kit, One-Color (Agilent Technologies, Santa Clara, CA, USA). Gene expression analysis was performed on samples from three independent experiments using a microarray (SurePrint G3 Human GE 8 × 60 K v2 Microarray; Agilent Technologies). The raw microarray data were deposited in the Gene Expression Omnibus (GEO; accession number, GSE62434; http://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc= GSE62434). CREB target genes were selected according to the CREB Target Gene Database (http://natural.salk.edu/CREB/), while NFAT target genes were selected by reference to Medline, as listed in Supplementary Table 1. Signal transduction pathways associated with each gene were identified according to the KEGG Pathway Database (http://www.genome.jp/kegg/pathway.html).

Quantitative real-time PCR.
To quantify mRNA levels, quantitative real-time PCR was carried out using TaqMan Probe and Premix Ex Taq (Probe qPCR; TaKaRa Bio Inc., Shiga, Japan) in a TaKaRa PCR Thermal Cycler Dice TP960 (TaKaRa Bio) according to the manufacturer's protocols. To normalize mRNA expression levels, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous internal control. Primers and probes used for RT-PCR are described in Table 2.
Scientific RepoRts | 6:18971 | DOI: 10.1038/srep18971 THP-1 cells (1 × 10 5 cells/mL) were treated with 10 mM AAPH for 3 h in the absence or presence of indicated concentrations of H 2 , and the NFAT translocation was investigated using immunofluorescence as described above.
Cell culture. THP-1 cells (ATCC) were cultured in RPMI1640 containing 10% FBS. Human aortic endothelial cells (HAEC) were obtained from Lonza and maintained in endothelial cell growth medium [EBM medium + growth supplements+ FCS (Lonza)]. Cells were cultured at 37 °C in a 5% CO 2 humidified atmosphere and were used for experiments from passage 4 to 8.
Statistical analysis. Statistical differences between groups were assessed by one-way analysis of variance (ANOVA) with Tukey-Kramer post hoc analysis unless otherwise mentioned. Statistical analyses were performed with IBM SPSS21 software. Results were considered significant at P < 0.05. When 0.01 < P < 0.05, the actual P values were noted. Data are presented as means ± standard deviations.