Molecular and Cellular Biochemistry

, Volume 403, Issue 1–2, pp 231–241 | Cite as

Simultaneous oral and inhalational intake of molecular hydrogen additively suppresses signaling pathways in rodents

  • Sayaka Sobue
  • Kazuaki Yamai
  • Mikako Ito
  • Kinji Ohno
  • Masafumi Ito
  • Takashi Iwamoto
  • Shanlou Qiao
  • Tetsuo Ohkuwa
  • Masatoshi Ichihara


Molecular hydrogen (H2) is an agent with potential applications in oxidative stress-related and/or inflammatory disorders. H2 is usually administered by inhaling H2-containing air (HCA) or by oral intake of H2-rich water (HRW). Despite mounting evidence, the molecular mechanism underlying the therapeutic effects and the optimal method of H2 administration remain unclear. Here, we investigated whether H2 affects signaling pathways and gene expression in a dosage- or dose regimen-dependent manner. We first examined the H2 concentrations in blood and organs after its administration and found that oral intake of HRW rapidly but transiently increased H2 concentrations in the liver and atrial blood, while H2 concentrations in arterial blood and the kidney were one-tenth of those in the liver and atrial blood. In contrast, inhalation of HCA increased H2 equally in both atrial and arterial blood. We next examined whether H2 alters gene expression in normal mouse livers using DNA microarray analysis after administration of HCA and HRW. Ingenuity Pathway Analysis revealed that H2 suppressed the expression of nuclear factor-kappa B (NF-κB)-regulated genes. Western blot analysis showed that H2 attenuated ERK, p38 MAPK, and NF-κB signaling in mouse livers. Finally, we evaluated whether the changes in gene expression were influenced by the route of H2 administration and found that the combination of both HRW and HCA had the most potent effects on signaling pathways and gene expression in systemic organs, suggesting that H2 may act not only through a dose-dependent mechanism but also through a complex molecular network.


Molecular hydrogen Gene expression NF-κB p38 ERK 



We thank Takae Hiraide, Taeko Nagano, and Ken-ichi Yoshida for technical assistance. This work was supported by Grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Supplementary material

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Supplementary material 1 (PDF 5260 kb)


  1. 1.
    Nakao A, Sugimoto R, Billiar TR, McCurry KR (2009) Therapeutic antioxidant medical gas. J Clin Biochem Nutr 44:1–13. doi: 10.3164/jcbn.08-193R CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, Katsura K, Katayama Y, Asoh S, Ohta S (2007) Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 13:688–694. doi: 10.1038/nm1577 CrossRefPubMedGoogle Scholar
  3. 3.
    Ohno K, Ito M, Ichihara M, Ito M (2012) Molecular hydrogen as an emerging therapeutic medical gas for neurodegenerative and other diseases. Oxid Med Cell Longev 2012:353152. doi: 10.1155/2012/353152 CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Ohta S (2014) Molecular hydrogen as a preventive and therapeutic medical gas: initiation, development and potential of hydrogen medicine. Pharmacol Ther. doi: 10.1016/j.pharmthera.2014.04.006 PubMedGoogle Scholar
  5. 5.
    Fukuda K, Asoh S, Ishikawa M, Yamamoto Y, Ohsawa I, Ohta S (2007) Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress. Biochem Biophys Res Commun 361:670–674. doi: 10.1016/j.bbrc.2007.07.088 CrossRefPubMedGoogle Scholar
  6. 6.
    Hayashida K, Sano M, Ohsawa I, Shinmura K, Tamaki K, Kimura K, Endo J, Katayama T, Kawamura A, Kohsaka S, Makino S, Ohta S, Ogawa S, Fukuda K (2008) Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia-reperfusion injury. Biochem Biophys Res Commun 373:30–35. doi: 10.1016/j.bbrc.2008.05.165 CrossRefPubMedGoogle Scholar
  7. 7.
    Fujita K, Seike T, Yutsudo N, Ohno M, Yamada H, Yamaguchi H, Sakumi K, Yamakawa Y, Kido MA, Takaki A, Katafuchi T, Tanaka Y, Nakabeppu Y, Noda M (2009) Hydrogen in drinking water reduces dopaminergic neuronal loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease. PLoS One 4:e7247. doi: 10.1371/journal.pone.0007247 CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Itoh T, Hamada N, Terazawa R, Ito M, Ohno K, Ichihara M, Nozawa Y, Ito M (2011) Molecular hydrogen inhibits lipopolysaccharide/interferon gamma-induced nitric oxide production through modulation of signal transduction in macrophages. Biochem Biophys Res Commun 411:143–149. doi: 10.1016/j.bbrc.2011.06.116 CrossRefPubMedGoogle Scholar
  9. 9.
    Xu XF, Zhang J (2013) Saturated hydrogen saline attenuates endotoxin-induced acute liver dysfunction in rats. Physiol Res 62:395–403PubMedGoogle Scholar
  10. 10.
    Cardinal JS, Zhan J, Wang Y, Sugimoto R, Tsung A, McCurry KR, Billiar TR, Nakao A (2010) Oral hydrogen water prevents chronic allograft nephropathy in rats. Kidney Int 77:101–109. doi: 10.1038/ki.2009.421 CrossRefPubMedGoogle Scholar
  11. 11.
    Kawamura T, Wakabayashi N, Shigemura N, Huang CS, Masutani K, Tanaka Y, Noda K, Peng X, Takahashi T, Billiar TR, Okumura M, Toyoda Y, Kensler TW, Nakao A (2013) Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo. Am J Physiol Lung Cell Mol Physiol. doi: 10.1152/ajplung.00164.2012 PubMedCentralPubMedGoogle Scholar
  12. 12.
    Ito M, Hirayama M, Yamai K, Goto S, Ito M, Ichihara M, Ohno K (2012) Drinking hydrogen water and intermittent hydrogen gas exposure, but not lactulose or continuous hydrogen gas exposure, prevent 6-hydorxydopamine-induced Parkinson’s disease in rats. Med Gas Res 2:15. doi: 10.1186/2045-9912-2-15 CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Sobue S, Murakami M, Banno Y, Ito H, Kimura A, Gao S, Furuhata A, Takagi A, Kojima T, Suzuki M, Nozawa Y, Murate T (2008) v-Src oncogene product increases sphingosine kinase 1 expression through mRNA stabilization: alteration of AU-rich element-binding proteins. Oncogene 27:6023–6033. doi: 10.1038/onc.2008.198 CrossRefPubMedGoogle Scholar
  14. 14.
    Ambade A, Catalano D, Lim A, Mandrekar P (2012) Inhibition of heat shock protein (molecular weight 90 kDa) attenuates proinflammatory cytokines and prevents lipopolysaccharide-induced liver injury in mice. Hepatology 55:1585–1595. doi: 10.1002/hep.24802 CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Kajiyama S, Hasegawa G, Asano M, Hosoda H, Fukui M, Nakamura N, Kitawaki J, Imai S, Nakano K, Ohta M, Adachi T, Obayashi H, Yoshikawa T (2008) Supplementation of hydrogen-rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance. Nutr Res 28:137–143. doi: 10.1016/j.nutres.2008.01.008 CrossRefPubMedGoogle Scholar
  16. 16.
    Kamimura N, Nishimaki K, Ohsawa I, Ohta S (2011) Molecular hydrogen improves obesity and diabetes by inducing hepatic FGF21 and stimulating energy metabolism in db/db mice. Obesity 19:1396–1403. doi: 10.1038/oby.2011.6 CrossRefPubMedGoogle Scholar
  17. 17.
  18. 18. Accessed 25 Jan 2015
  19. 19.
    Pahl HL (1999) Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18:6853–6866. doi: 10.1038/sj.onc.1203239 CrossRefPubMedGoogle Scholar
  20. 20.
    Feuerhake F, Kutok JL, Monti S, Chen W, LaCasce AS, Cattoretti G, Kurtin P, Pinkus GS, de Leval L, Harris NL, Savage KJ, Neuberg D, Habermann TM, Dalla-Favera R, Golub TR, Aster JC, Shipp MA (2005) NFkappaB activity, function, and target-gene signatures in primary mediastinal large B-cell lymphoma and diffuse large B-cell lymphoma subtypes. Blood 106:1392–1399. doi: 10.1182/blood-2004-12-4901 CrossRefPubMedGoogle Scholar
  21. 21.
    Bunting K, Rao S, Hardy K, Woltring D, Denyer GS, Wang J, Gerondakis S, Shannon MF (2007) Genome-wide analysis of gene expression in T cells to identify targets of the NF-kappa B transcription factor c-Rel. J Immunol 178:7097–7109CrossRefPubMedGoogle Scholar
  22. 22.
    Naamane N, van Helden J, Eizirik DL (2007) In silico identification of NF-kappaB-regulated genes in pancreatic beta-cells. BMC Bioinform 8:55. doi: 10.1186/1471-2105-8-55 CrossRefGoogle Scholar
  23. 23.
    Takase O, Marumo T, Hishikawa K, Fujita T, Quigg RJ, Hayashi M (2008) NF-kappaB-dependent genes induced by proteinuria and identified using DNA microarrays. Clin Exp Nephrol 12:181–188. doi: 10.1007/s10157-008-0038-5 CrossRefPubMedGoogle Scholar
  24. 24.
    Nagata K, Nakashima-Kamimura N, Mikami T, Ohsawa I, Ohta S (2009) Consumption of molecular hydrogen prevents the stress-induced impairments in hippocampus-dependent learning tasks during chronic physical restraint in mice. Neuropsychopharmacology 34:501–508. doi: 10.1038/npp.2008.95 CrossRefPubMedGoogle Scholar
  25. 25.
    Liu C, Kurokawa R, Fujino M, Hirano S, Sato B, Li XK (2014) Estimation of the hydrogen concentration in rat tissue using an airtight tube following the administration of hydrogen via various routes. Sci Rep 4:5485. doi: 10.1038/srep05485 PubMedCentralPubMedGoogle Scholar
  26. 26.
    Nakai Y, Sato B, Ushiama S, Okada S, Abe K, Arai S (2011) Hepatic oxidoreduction-related genes are upregulated by administration of hydrogen-saturated drinking water. Biosci Biotechnol Biochem 75:774–776CrossRefPubMedGoogle Scholar
  27. 27.
    Luedde T, Beraza N, Kotsikoris V, van Loo G, Nenci A, De Vos R, Roskams T, Trautwein C, Pasparakis M (2007) Deletion of NEMO/IKKgamma in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma. Cancer Cell 11:119–132. doi: 10.1016/j.ccr.2006.12.016 CrossRefPubMedGoogle Scholar
  28. 28.
    Campbell JS, Argast GM, Yuen SY, Hayes B, Fausto N (2011) Inactivation of p38 MAPK during liver regeneration. Int J Biochem Cell Biol 43:180–188. doi: 10.1016/j.biocel.2010.08.002 CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Imai J, Katagiri H, Yamada T, Ishigaki Y, Suzuki T, Kudo H, Uno K, Hasegawa Y, Gao J, Kaneko K, Ishihara H, Niijima A, Nakazato M, Asano T, Minokoshi Y, Oka Y (2008) Regulation of pancreatic beta cell mass by neuronal signals from the liver. Science 322:1250–1254. doi: 10.1126/science.1163971 CrossRefPubMedGoogle Scholar
  30. 30.
    Matsumoto A, Yamafuji M, Tachibana T, Nakabeppu Y, Noda M, Nakaya H (2013) Oral ‘hydrogen water’ induces neuroprotective ghrelin secretion in mice. Sci Rep 3:3273. doi: 10.1038/srep03273 CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sayaka Sobue
    • 1
  • Kazuaki Yamai
    • 1
    • 2
  • Mikako Ito
    • 3
  • Kinji Ohno
    • 3
  • Masafumi Ito
    • 4
  • Takashi Iwamoto
    • 1
  • Shanlou Qiao
    • 1
  • Tetsuo Ohkuwa
    • 2
  • Masatoshi Ichihara
    • 1
  1. 1.Department of Biomedical Sciences, College of Life and Health SciencesChubu UniversityKasugaiJapan
  2. 2.Department of Materials Science and EngineeringNagoya Institute of Technology, Graduate School of EngineeringNagoyaJapan
  3. 3.Division of Neurogenetics, Center for Neurological Diseases and CancerNagoya University Graduate School of MedicineNagoyaJapan
  4. 4.Research Team for Mechanism of AgingTokyo Metropolitan Institute of GerontologyTokyoJapan

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