{"id":27564,"date":"2024-01-03T21:48:44","date_gmt":"2024-01-03T19:48:44","guid":{"rendered":"https:\/\/hho-bulgaria.com\/h2o2-promotes-ghrelin-secretion-for-neuroprotection\/"},"modified":"2024-01-29T21:29:50","modified_gmt":"2024-01-29T19:29:50","slug":"h2o2-promotes-ghrelin-secretion-for-neuroprotection","status":"publish","type":"post","link":"https:\/\/hho-bulgaria.com\/en\/h2o2-promotes-ghrelin-secretion-for-neuroprotection\/","title":{"rendered":"H2O2 promotes ghrelin secretion for neuroprotection"},"content":{"rendered":"<div class=\"jig-ncbiinpagenav\" data-jigconfig=\"smoothScroll: false, allHeadingLevels: ['h2'], headingExclude: ':hidden,.nomenu'\" id=\"ui-ncbiinpagenav-1\">\n<div class=\"fm-sec half_rhythm no_top_margin\">\n<div class=\"fm-flexbox\">\n<div class=\"fm-citation\">\n<div class=\"citation-default\">\n<div class=\"part1\"><span role=\"menubar\"><a href=\"#\" role=\"menuitem\" aria-expanded=\"false\" aria-haspopup=\"true\">Sci Rep.<\/a><\/span> 2013; 3: 3273. <\/div>\n<div class=\"part2\"><span class=\"fm-vol-iss-date\">Published online 2013 Nov 20. <\/span>  <span class=\"doi\"><span>doi:&nbsp;<\/span><a href=\"\/\/doi.org\/10.1038%2Fsrep03273\" target=\"_blank\" rel=\"noopener noreferrer\" ref=\"reftype=other&amp;article-id=4070541&amp;issue-id=217774&amp;journal-id=1579&amp;FROM=Article%7CFront%20Matter&amp;TO=Content%20Provider%7CCrosslink%7CDOI\">10.1038\/srep03273<\/a><\/span><\/div>\n<\/div>\n<\/div>\n<div class=\"fm-ids\">\n<div class=\"fm-citation-pmcid\"><span class=\"fm-citation-ids-label\">PMCID: <\/span><span>PMC4070541<\/span><\/div>\n<div class=\"fm-citation-pmid\">PMID: <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/24253616\">24253616<\/a><\/div>\n<\/div>\n<\/div>\n<h1 class=\"content-title\">Oral \u2018hydrogen water&#8217; induces neuroprotective ghrelin secretion in mice<\/h1>\n<div class=\"half_rhythm\">\n<div class=\"contrib-group fm-author\"><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Matsumoto%20A%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139809139277632\" co-class=\"co-affbox\">Akio Matsumoto<\/a>,<sup>a,<\/sup><sup>1,<\/sup><sup>2,<\/sup><sup>5,<\/sup><sup>6<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Yamafuji%20M%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139809139274416\" co-class=\"co-affbox\">Megumi Yamafuji<\/a>,<sup>3,<\/sup><sup>6<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Tachibana%20T%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139809139271696\" co-class=\"co-affbox\">Tomoko Tachibana<\/a>,<sup>1<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Nakabeppu%20Y%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139809139269712\" co-class=\"co-affbox\">Yusaku Nakabeppu<\/a>,<sup>4,<\/sup><sup>5<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Noda%20M%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139809180800544\" co-class=\"co-affbox\">Mami Noda<\/a>,<sup>3,<\/sup><sup>5<\/sup> and  <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Nakaya%20H%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139809180797824\" co-class=\"co-affbox\">Haruaki Nakaya<\/a><sup>1<\/sup><\/div>\n<div style=\"display:none\" class=\"contrib-group aff-tip\">\n<div id=\"_co_idm139809139277632\">\n<h3 class=\"no_margin\">Akio Matsumoto<\/h3>\n<p><sup>1<\/sup>Department of Pharmacology, Graduate School of Medicine, Chiba University, Chiba<\/p>\n<p><sup>2<\/sup>Division of Molecular Design, Medical Institute of Bioregulation, Kyushu University, Fukuoka<\/p>\n<p><sup>5<\/sup>Research Center for Nucleotide Pool, Kyushu University, Fukuoka, Japan<\/p>\n<p><sup>6<\/sup>These authors contributed equally to this work.<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Matsumoto%20A%5BAuthor%5D\">Akio Matsumoto<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139809139274416\">\n<h3 class=\"no_margin\">Megumi Yamafuji<\/h3>\n<p><sup>3<\/sup>Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka<\/p>\n<p><sup>6<\/sup>These authors contributed equally to this work.<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Yamafuji%20M%5BAuthor%5D\">Megumi Yamafuji<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139809139271696\">\n<h3 class=\"no_margin\">Tomoko Tachibana<\/h3>\n<p><sup>1<\/sup>Department of Pharmacology, Graduate School of Medicine, Chiba University, Chiba<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Tachibana%20T%5BAuthor%5D\">Tomoko Tachibana<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139809139269712\">\n<h3 class=\"no_margin\">Yusaku Nakabeppu<\/h3>\n<p><sup>4<\/sup>Division of Neurofunctional Genomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka<\/p>\n<p><sup>5<\/sup>Research Center for Nucleotide Pool, Kyushu University, Fukuoka, Japan<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Nakabeppu%20Y%5BAuthor%5D\">Yusaku Nakabeppu<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139809180800544\">\n<h3 class=\"no_margin\">Mami Noda<\/h3>\n<p><sup>3<\/sup>Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka<\/p>\n<p><sup>5<\/sup>Research Center for Nucleotide Pool, Kyushu University, Fukuoka, Japan<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Noda%20M%5BAuthor%5D\">Mami Noda<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139809180797824\">\n<h3 class=\"no_margin\">Haruaki Nakaya<\/h3>\n<p><sup>1<\/sup>Department of Pharmacology, Graduate School of Medicine, Chiba University, Chiba<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Nakaya%20H%5BAuthor%5D\">Haruaki Nakaya<\/a><\/div>\n<\/div>\n<\/div>\n<\/div>\n<div class=\"half_rhythm\">\n<div class=\"togglers fm-copyright-license\"><a href=\"#\" class=\"pmctoggle\" rid=\"idm139809140825760_ai\">Author information<\/a> <a href=\"#\" class=\"pmctoggle\" rid=\"idm139809140825760_an\">Article notes<\/a> <a href=\"#\" class=\"pmctoggle\" rid=\"idm139809140825760_cpl\">Copyright and License information<\/a> <a href=\"\/pmc\/about\/disclaimer\/\" style=\"margin-left: 1em\">PMC Disclaimer<\/a><\/div>\n<div class=\"fm-authors-info hide half_rhythm\" id=\"idm139809140825760_ai\" style=\"display:none\">\n<div class=\"fm-affl\" id=\"a1\"><sup>1<\/sup>Department of Pharmacology, Graduate School of Medicine, Chiba University, Chiba<\/div>\n<div class=\"fm-affl\" id=\"a2\"><sup>2<\/sup>Division of Molecular Design, Medical Institute of Bioregulation, Kyushu University, Fukuoka<\/div>\n<div class=\"fm-affl\" id=\"a3\"><sup>3<\/sup>Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka<\/div>\n<div class=\"fm-affl\" id=\"a4\"><sup>4<\/sup>Division of Neurofunctional Genomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka<\/div>\n<div class=\"fm-affl\" id=\"a5\"><sup>5<\/sup>Research Center for Nucleotide Pool, Kyushu University, Fukuoka, Japan<\/div>\n<div class=\"fm-affl\" id=\"a6\"><sup>6<\/sup>These authors contributed equally to this work.<\/div>\n<div id=\"c1\"><sup>a<\/sup><span class=\"email-label\">Email: <\/span><a href=\"mailto:dev@null\" data-email=\"pj.u-abihc.ytlucaf@oika\" class=\"oemail\">pj.u-abihc.ytlucaf@oika<\/a><\/div>\n<\/div>\n<div class=\"fm-article-notes hide half_rhythm\" id=\"idm139809140825760_an\" style=\"display:none\">\n<div class=\"fm-pubdate half_rhythm\">Received 2013 Jul 2; Accepted 2013 Nov 1.<\/div>\n<\/div>\n<div class=\"permissions half_rhythm hide\" id=\"idm139809140825760_cpl\" style=\"display:none\">\n<div class=\"fm-copyright half_rhythm\"><a href=\"\/pmc\/about\/copyright\/\">Copyright<\/a>  \u00a9 2013, Macmillan Publishers Limited. All rights reserved<\/div>\n<div class=\"license half_rhythm\">This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit <a href=\"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/3.0\/\" data-ga-action=\"click_feat_suppl\" ref=\"reftype=extlink&amp;article-id=4070541&amp;issue-id=217774&amp;journal-id=1579&amp;FROM=Article%7CFront%20Matter&amp;TO=External%7CLink%7CURI\" target=\"_blank\" rel=\"noopener\">http:\/\/creativecommons.org\/licenses\/by-nc-nd\/3.0\/<\/a><\/div>\n<\/div>\n<\/div>\n<div id=\"pmclinksbox\" class=\"links-box whole_rhythm hidden\" role=\"complementary\" aria-label=\"Related or updated information about this article.\"><\/div>\n<\/div>\n<div class=\"sec\"><\/div>\n<div id=\"abstract-a.h.b.n\" lang=\"en\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><span role=\"menubar\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"menuitem\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/span><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"abstract-a.h.b.ntitle\">Abstract<\/h2>\n<p><!--article-meta--><\/p>\n<div>\n<p class=\"p p-first-last\">The therapeutic potential of molecular hydrogen (H<sub>2<\/sub>) is emerging in a number of human diseases and in their animal models, including in particular Parkinson&#8217;s disease (PD). H<sub>2<\/sub> supplementation of drinking water has been shown to exert disease-modifying effects in PD patients and neuroprotective effects in experimental PD model mice. However, H<sub>2<\/sub> supplementation does not result in detectable changes in striatal H<sub>2<\/sub> levels, indicating an indirect effect. Here we show that H<sub>2<\/sub> supplementation increases gastric expression of mRNA encoding ghrelin, a growth hormone secretagogue, and ghrelin secretion, which are antagonized by the \u03b2<sub>1<\/sub>-adrenoceptor blocker, atenolol. Strikingly, the neuroprotective effect of H<sub>2<\/sub> water was abolished by either administration of the ghrelin receptor-antagonist, D-Lys<sup>3<\/sup> GHRP-6, or atenolol. Thus, the neuroprotective effect of H<sub>2<\/sub> in PD is mediated by enhanced production of ghrelin. Our findings point to potential, novel strategies for ameliorating pathophysiology in which a protective effect of H<sub>2<\/sub> supplementation has been demonstrated.<\/p>\n<\/div>\n<\/div>\n<div id=\"body-a.i\" class=\"tsec sec\">\n<h2 class=\"headless nomenu\"><\/h2>\n<p class=\"p p-first\">Therapeutic applications of molecular hydrogen (H<sub>2<\/sub>) have been reported in a variety of human diseases and their animal models<sup><a href=\"#b1\" rid=\"b1\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">1<\/a><\/sup>, including ischemia-reperfusion injury<sup><a href=\"#b2\" rid=\"b2\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">2<\/a><\/sup><sup>,<a href=\"#b3\" rid=\"b3\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">3<\/a><\/sup><sup>,<a href=\"#b4\" rid=\"b4\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">4<\/a><\/sup>, metabolic syndrome<sup><a href=\"#b5\" rid=\"b5\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">5<\/a><\/sup>, diabetes mellitus type 2<sup><a href=\"#b6\" rid=\"b6\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">6<\/a><\/sup>, organ transplantation<sup><a href=\"#b7\" rid=\"b7\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">7<\/a><\/sup><sup>,<a href=\"#b8\" rid=\"b8\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">8<\/a><\/sup><sup>,<a href=\"#b9\" rid=\"b9\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">9<\/a><\/sup>, reduction of adverse effects of anti-tumor drug therapy<sup><a href=\"#b10\" rid=\"b10\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">10<\/a><\/sup><sup>,<a href=\"#b11\" rid=\"b11\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">11<\/a><\/sup> and radiation therapy<sup><a href=\"#b12\" rid=\"b12\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">12<\/a><\/sup><sup>,<a href=\"#b13\" rid=\"b13\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">13<\/a><\/sup>. Although the mechanism of action of H<sub>2<\/sub> has not been clearly demonstrated, it is assumed that its anti-oxidative properties, particularly against hydroxyl radical (\u00b7OH) and peroxinitrite (ONOO<sup>\u2212<\/sup>), are likely to underlie therapeutic efficacy<sup><a href=\"#b2\" rid=\"b2\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">2<\/a><\/sup>. Unlike other medical-gas therapy, H<sub>2<\/sub> can be applied in air for inhalation or in solution for drinking, intravenous injection or dialysis. Whereas intravenous injection or dialysis delivers H<sub>2<\/sub> directly into the blood stream, oral hydrogen-supplemented water (hydrogen water, H<sub>2<\/sub>-water) must be absorbed into the circulation resulting in limited H<sub>2<\/sub> concentrations in the blood and in target organs<sup><a href=\"#b7\" rid=\"b7\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">7<\/a><\/sup><sup>,<a href=\"#b14\" rid=\"b14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a><\/sup>.<\/p>\n<p>Parkinson&#8217;s disease (PD) has been a major focus in the field of oxidative stress and disease, because it is thought that degeneration of dopaminergic neurons can be triggered and aggravated by the accumulation of oxidative damage. However, although antioxidant therapies have been assessed in PD patients, clinical efficacy has not been established<sup><a href=\"#b15\" rid=\"b15\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">15<\/a><\/sup><sup>,<a href=\"#b16\" rid=\"b16\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">16<\/a><\/sup>. In contrast, a pilot study of hydrogen water therapy in PD patients has shown promising results<sup><a href=\"#b17\" rid=\"b17\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">17<\/a><\/sup>, and it has been reported that hydrogen water exhibits neuroprotective effects<sup><a href=\"#b14\" rid=\"b14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a><\/sup> in the murine MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced PD model<sup><a href=\"#b18\" rid=\"b18\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">18<\/a><\/sup>. H<sub>2<\/sub> levels were below measurable thresholds in the substantia nigra in PD model mice<sup><a href=\"#b14\" rid=\"b14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a><\/sup>, and hydrogen water, but not continuous inhalation of 2% H<sub>2<\/sub>, prevented the development of PD in a rat model<sup><a href=\"#b19\" rid=\"b19\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">19<\/a><\/sup>. These findings suggest that the therapeutic effects of hydrogen water may not require its anti-oxidant activity in the brain, and further that its efficacy requires processing that is consequent upon oral administration.<\/p>\n<p class=\"p\">The purpose of the present study was to employ PD model mice to elucidate the underlying mechanism of the neuroprotective effects of oral H<sub>2<\/sub>-water. In particular, we hypothesized that oral H<sub>2<\/sub> induces a messenger molecule, which travels to the brain and exerts neuroprotective activity.<\/p>\n<\/div>\n<div id=\"sec-a.i.d\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"sec-a.i.dtitle\">Results<\/h2>\n<div id=\"sec-a.i.d.b\" class=\"sec sec-first\">\n<h3 id=\"sec-a.i.d.btitle\">Oral hydrogen water induces ghrelin gene expression in the stomach<\/h3>\n<p class=\"p p-first\">The stomach functions as an endocrine organ that secretes various peptide hormones with a broad range of physiological effects. We first focused on the stomach to investigate possible effects of oral H<sub>2<\/sub>-water at the level of gene induction. In a previous study<sup><a href=\"#b14\" rid=\"b14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a><\/sup>, it was reported that drinking H<sub>2<\/sub>-water for a period of 7 days prior to MPTP injection protected against MPTP toxicity. We administered oral H<sub>2<\/sub>-water for 4 consecutive days and analyzed expression in stomach tissue of gastrin, somatostatin, and ghrelin by real-time PCR method. Levels of ghrelin mRNA increased 1.9-fold in H<sub>2<\/sub>-water-treated versus control mice (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f1\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"f1\" rid-ob=\"ob-f1\" co-legend-rid=\"lgnd_f1\" rel=\"noopener\"><span>Figure 1<\/span><\/a>), whereas no effects of hydrogen water were detected on expression of the somatostatin gene (expressed only at trace levels) or the gastrin gene (expression of which was highly variable from individual to individual) (not shown).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"f1\" co-legend-rid=\"lgnd_f1\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f1\/\" target=\"figure\" rid-figpopup=\"f1\" rid-ob=\"ob-f1\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139809144552816\"><img decoding=\"async\" loading=\"lazy\" class=\"fig-image\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is srep03273-f1.jpg\" title=\"An external file that holds a picture, illustration, etc.\nObject name is srep03273-f1.jpg\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/srep03273-f1.jpg\"><\/div>\n<p><\/a><\/p>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139809144552816\" style=\"display: none;\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f1\/\" target=\"figure\" rid-figpopup=\"f1\" rid-ob=\"ob-f1\" rel=\"noopener\"><\/a><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f1\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_f1\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f1\/\" target=\"figure\" rid-figpopup=\"f1\" rid-ob=\"ob-f1\" rel=\"noopener\">Figure 1<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\"><!--caption a8--><strong>Oral H<sub>2<\/sub> water increases ghrelin gene expression in the mouse stomach.<\/strong><\/p>\n<p>Control water, or H<sub>2<\/sub> water (0.8\u2005ml\/mouse) made with a stick of magnesium (about 0.04\u2005mM H<sub>2<\/sub&gt;; see Methods), was administered to mice (41\u201348 weeks of age; n = 4 per group) once a day for 4 days. Three hr after the final ingestion, the stomach was removed and prepared for qPCR. Data were normalized with respect to expression of RPL4 mRNA and are represented as mean \u00b1 SEM.<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p class=\"p p-last\">To examine the time-course of ghrelin induction by hydrogen water, mice were administered hydrogen water or control water once a day for 0 (control) 1, 2 or 4 days and ghrelin levels were measured by ELISA in plasma derived from blood obtained 4\u20135\u2005hours after the final administration of H<sub>2<\/sub>&#8211; or control water. Only mice that received H<sub>2<\/sub> water for 4-day exhibited a significant increase in plasma ghrelin level, although mice administered hydrogen water for 2-day showed a non-significant increase (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f2\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"f2\" rid-ob=\"ob-f2\" co-legend-rid=\"lgnd_f2\" rel=\"noopener\"><span>Figure 2<\/span><\/a>).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"f2\" co-legend-rid=\"lgnd_f2\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f2\/\" target=\"figure\" rid-figpopup=\"f2\" rid-ob=\"ob-f2\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139809144547648\"><img decoding=\"async\" loading=\"lazy\" class=\"fig-image\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is srep03273-f2.jpg\" title=\"An external file that holds a picture, illustration, etc.\nObject name is srep03273-f2.jpg\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/srep03273-f2.jpg\"><\/div>\n<p><\/a><\/p>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139809144547648\" style=\"display: none;\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f2\/\" target=\"figure\" rid-figpopup=\"f2\" rid-ob=\"ob-f2\" rel=\"noopener\"><\/a><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f2\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_f2\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f2\/\" target=\"figure\" rid-figpopup=\"f2\" rid-ob=\"ob-f2\" rel=\"noopener\">Figure 2<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\"><!--caption a8--><strong>Plasma ghrelin levels following administration of oral H<sub>2<\/sub> water.<\/strong><\/p>\n<p>Mice (16\u201321 weeks, average 18.5 weeks, n = 5\u201310) received H<sub>2<\/sub> water (or control water) as in <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f1\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"f1\" rid-ob=\"ob-f1\" co-legend-rid=\"lgnd_f1\" rel=\"noopener\"><span>Fig. 1<\/span><\/a> for 1, 2 or 4\u2005d. Blood was collected on the final day of the experiment, 4\u2005hr after the last ingestion, and plasma levels of ghrelin were quantified by ELISA. Panel shows data from male mice. The ghrelin level was significantly higher only in the group of mice drinking H<sub>2<\/sub> water for 4\u2005d. Data are represented as mean \u00b1 SEM.<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"sec-a.i.d.c\" class=\"sec\">\n<h3 id=\"sec-a.i.d.ctitle\">\u03b2<sub>1<\/sub>-adrenergic receptor signaling mediates enhancement of ghrelin secretion by oral hydrogen water<\/h3>\n<p class=\"p p-first-last\">It has been shown that gastric secretion of ghrelin is regulated by local environmental cues including blood glucose, estrogen, insulin and catecholamines<sup><a href=\"#b20\" rid=\"b20\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">20<\/a><\/sup><sup>,<a href=\"#b21\" rid=\"b21\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">21<\/a><\/sup><sup>,<a href=\"#b22\" rid=\"b22\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">22<\/a><\/sup>. In particular, it has been reported that \u03b2<sub>1<\/sub>-adrenergic receptor stimulation increases ghrelin secretion <em>in vitro<\/em> and <em>in vivo<\/em><sup><a href=\"#b22\" rid=\"b22\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">22<\/a><\/sup><sup>,<a href=\"#b23\" rid=\"b23\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">23<\/a><\/sup>. We verified the expression of \u03b2<sub>1<\/sub>&#8211; and \u03b2<sub>2<\/sub>&#8211; adrenergic receptors in stomach tissue samples by real-time PCR method, and determined that there were no significant changes in the levels of expression after administration of oral H<sub>2<\/sub>-water for 4 days (data not shown). The increase in plasma ghrelin levels by oral hydrogen water was eliminated by administration of the \u03b2<sub>1<\/sub>-adrenergic receptor-specific blocker, atenolol (10\u2005mg\/kg i.p.) injected 30\u2005min prior to H<sub>2<\/sub> water administration on each of four days (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"f3\" rid-ob=\"ob-f3\" co-legend-rid=\"lgnd_f3\" rel=\"noopener\"><span>Figure 3<\/span><\/a>). Thus, activation of \u03b2<sub>1<\/sub>-adrenergic receptors is required for hydrogen water-induced enhancement of circulating ghrelin.<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"f3\" co-legend-rid=\"lgnd_f3\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f3\/\" target=\"figure\" rid-figpopup=\"f3\" rid-ob=\"ob-f3\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139809144542896\"><img decoding=\"async\" loading=\"lazy\" class=\"fig-image\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is srep03273-f3.jpg\" title=\"An external file that holds a picture, illustration, etc.\nObject name is srep03273-f3.jpg\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/srep03273-f3.jpg\"><\/div>\n<p><\/a><\/p>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139809144542896\" style=\"display: none;\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f3\/\" target=\"figure\" rid-figpopup=\"f3\" rid-ob=\"ob-f3\" rel=\"noopener\"><\/a><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f3\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_f3\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f3\/\" target=\"figure\" rid-figpopup=\"f3\" rid-ob=\"ob-f3\" rel=\"noopener\">Figure 3<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\"><!--caption a8--><strong>H<sub>2<\/sub> water increases plasma ghrelin levels in a \u03b2<sub>1<\/sub>-adrenergic receptor-dependent manner.<\/strong><\/p>\n<p>Plasma ghrelin levels were measured on the last day of experiment. Each group of mice (11\u201313 weeks of age; n = 4\u20135 for each group) was administered control water or H<sub>2<\/sub> water (made with an open-air water electrolysis system) once a day for 4\u2005d with or without i.p. injection of atenolol (10\u2005mg\/kg) prior to water ingestion. The H<sub>2<\/sub> water group showed a significant increase in plasma ghrelin level compared to the control group (* p = 0.038), which was abrogated by pretreatment with atenolol (** p = 0.039). Datafare represented as mean \u00b1 SEM.<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"sec-a.i.d.d\" class=\"sec sec-last\">\n<h3 id=\"sec-a.i.d.dtitle\">Blockades of ghrelin action abrogates the protective effect of H<sub>2<\/sub> water in PD model mice<\/h3>\n<p class=\"p p-first-last\">In a previous report, oral H<sub>2<\/sub>-water exhibited significant protective effects in MPTP-induced PD model mice against the loss of dopaminergic neurons from the substantia nigra<sup><a href=\"#b14\" rid=\"b14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a><\/sup>. To test directly the role of ghrelin, either the growth hormone secretagogue receptor antagonist, D-Lys<sup>3<\/sup> GHRP-6 (100\u2005nmol\/day i.p.), or \u03b2<sub>1<\/sub>-adrenoceptor blocker, atenolol (10\u2005mg\/kg i.p.), was administered along with control or hydrogen water for 7 days prior to administration of MPTP. Loss of dopaminergic neurons from the substantia nigra was evaluated seven days following administration of MPTP. As shown in <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f4\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"f4\" rid-ob=\"ob-f4\" co-legend-rid=\"lgnd_f4\" rel=\"noopener\"><span>Figure 4<\/span><\/a>, systemic administration of MPTP caused a significant loss of dopaminergic neurons from the pars compacta of the substantia nigra (SNpc) as assessed by immunohistochemical detection of tyrosine hydroxylase (TH) (a), and further confirmed by immunological detection of TH protein in the substantia nigra with actin as control (b), and stereological analysis (c). Whereas administration of H<sub>2<\/sub> water alone in control mice had no effect, the loss of dopaminergic neurons in MPTP-treated mice was significantly decreased by administration of hydrogen water, as previously reported<sup><a href=\"#b14\" rid=\"b14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a><\/sup>. Strikingly, although administration of D-Lys<sup>3<\/sup> GHRP-6 or atenolol in control mice had no effect alone and D-Lys<sup>3<\/sup> GHRP-6 or atenolol did not affect MPTP-induced loss of dopaminergic neurons, the protective effects of hydrogen water were eliminated by either one of D-Lys<sup>3<\/sup> GHRP-6 or atenolol. Thus, induction of gastric ghrelin production and subsequent activation of ghrelin-initiated signal transduction underlies the protective effects of hydrogen water in the MPTP model of PD.<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"f4\" co-legend-rid=\"lgnd_f4\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f4\/\" target=\"figure\" rid-figpopup=\"f4\" rid-ob=\"ob-f4\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139809137750080\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f4\/\" target=\"figure\" rid-figpopup=\"f4\" rid-ob=\"ob-f4\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=4070541_srep03273-f4.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is srep03273-f4.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/srep03273-f4.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139809137750080\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f4\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_f4\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/figure\/f4\/\" target=\"figure\" rid-figpopup=\"f4\" rid-ob=\"ob-f4\" rel=\"noopener\">Figure 4<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\"><!--caption a8--><strong>Inhibition of ghrelin secretion or the ghrelin receptor antagonist cancels the neuroprotective effect of oral H<sub>2<\/sub> water in MPTP-induced Parkinson&#8217;s disease model mice (male, 8\u201312 weeks, n = 2\u20135 for each group).<\/strong><\/p>\n<p>Saturated H<sub>2<\/sub> water was made daily as described in materials and methods. (a): Tyrosine hydroxylase (TH) staining was performed in substantia nigra pars compacta from saline-injected (i\u2013iv), D-Lys<sup>3<\/sup> GHRP-6-injected (v\u2013viii), and atenolol-injected (ix\u2013xii) mice, supplied with either normal tap water (control) or H<sub>2<\/sub> water for 7 days. MPTP was injected (i.p.) after 7 days of D-Lys<sup>3<\/sup> GHRP-6, atenolol, or saline-injection (ii, iv, vi, viii, x, and xii). (b): Summary of the immunoblotting analysis of TH protein in the substantia nigra tissue (n = 3\u20136 for each group) and the representative blot images were shown underneath (two samples per group). The TH band intensity was normalized to the band of actin on the same sample. (## p = 0.007, i vs. ii; 0.010, ii vs. iv; 0.004, vi vs. vii; # p = 0.043, iv vs. viii; 0.023, v vs. vi). Data in figures are represented as mean \u00b1 SEM. (c): Summary of the stereological analysis of nigral dopaminergic neurons. Although MPTP injections caused significant loss of TH-positive neurons (ii) (** p = 0.0001, i vs. ii), drinking H<sub>2<\/sub> water for 7 days prior to MPTP-treatment significantly attenuates the loss of TH-positive cells (iv) (# p = 0.008, ii vs. iv). D-Lys<sup>3<\/sup> GHRP-6, growth hormone secretagogue receptor antagonist, or \u03b2<sub>1<\/sub>-adrenoceptor blocker, atenolol, canceled the preservation effect of oral H<sub>2<\/sub> water (viii and xii) (# p = 0.008; ii vs. iv; p = 0.003, iv vs. viii; p = 0.0003, iv vs. xii). Data in figures are represented as mean \u00b1 SEM. The statistical significance of data was assessed by one-way ANOVA followed by Benferroni test.<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"sec-a.i.e\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"sec-a.i.etitle\">Discussion<\/h2>\n<p class=\"p p-first\">Our findings demonstrate that the neuroprotective effects of oral hydrogen water, which produces negligible levels of H<sub>2<\/sub> in the brain, result from gastric induction of the neuroprotective peptide hormone ghrelin and the subsequent activation of ghrelin receptors. In addition, we have shown an obligate role for \u03b2<sub>1<\/sub>-adrenergic receptors in hydrogen water-induced ghrelin up-regulation in plasma, consistent with previous reports that adrenergic stimulation regulates ghrelin release <em>in vitro<\/em> and <em>in vivo<\/em><sup><a href=\"#b22\" rid=\"b22\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">22<\/a><\/sup><sup>,<a href=\"#b24\" rid=\"b24\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">24<\/a><\/sup><sup>,<a href=\"#b25\" rid=\"b25\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">25<\/a><\/sup>.<\/p>\n<p>The neuroprotective effects of ghrelin in PD are well-established<sup><a href=\"#b26\" rid=\"b26\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">26<\/a><\/sup>, and it has been demonstrated that the receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), is highly expressed by dopaminergic neurons of the substantia nigra<sup><a href=\"#b27\" rid=\"b27\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">27<\/a><\/sup>. It has been suggested that ghrelin protects nigrostriatal dopamine neurons via an uncoupling protein 2 (UCP2)-dependent mitochondrial mechanism<sup><a href=\"#b28\" rid=\"b28\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">28<\/a><\/sup><sup>,<a href=\"#b29\" rid=\"b29\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">29<\/a><\/sup>. However, we found that the expression of neither UCP2 mRNA nor protein was significantly upregulated by administration of H<sub>2<\/sub> water drinking for 7 days (data not shown). This finding suggests an alternative signaling mechanism downstream of GHSR activation, perhaps involving PI3K\/Akt<sup><a href=\"#b30\" rid=\"b30\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">30<\/a><\/sup>.<\/p>\n<p class=\"p p-last\">It was reported that administration of saturated hydrogen water (approx. 0.8\u2005mM) led to symptomatic improvement in PD patients<sup><a href=\"#b17\" rid=\"b17\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">17<\/a><\/sup>. Administration of hydrogen water at about 0.05% saturation successfully maintained dopaminergic neurons in MPTP-induced PD model mice<sup><a href=\"#b14\" rid=\"b14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a><\/sup>. We employed three different methods to prepare hydrogen water (see Methods), which resulted in H<sub>2<\/sub> concentration of 0.04\u20130.8\u2005mM, and we observed that the effects of hydrogen water on ghrelin induction and protection of dopamine neurons were dose-independent over this range. Thus, small amounts of oral H<sub>2<\/sub> are sufficient for gastric induction of ghrelin and subsequent neuroprotection, in the absence of detectable H<sub>2<\/sub> in the brain. Interestingly, gut microbes can produce molecular hydrogen constitutively and lactulose, a synthetic disaccharide, is an effective substrate to enhance bacterial H<sub>2<\/sub> production in the colon<sup><a href=\"#b31\" rid=\"b31\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">31<\/a><\/sup>. However, ingestion of lactulose had no significant effect on dopaminergic neuron survival in 6-OHDA-induced PD model rats (although alveolar H<sub>2<\/sub> concentrations were elevated)<sup><a href=\"#b19\" rid=\"b19\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">19<\/a><\/sup>. These results emphasize the importance of gastric ghrelin induction in the neuroprotective action of H<sub>2<\/sub>. Oral H<sub>2<\/sub>-water is being explored as a therapeutic for PD as well as a variety of other human pathophysiological conditions<sup><a href=\"#b17\" rid=\"b17\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">17<\/a><\/sup><sup>,<a href=\"#b32\" rid=\"b32\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">32<\/a><\/sup><sup>,<a href=\"#b33\" rid=\"b33\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">33<\/a><\/sup> under the generally held assumption that the mechanism of action of supplemental H<sub>2<\/sub> is likely to reflect an antioxidative role. Our findings that oral H<sub>2<\/sub> water exerts a neuroprotective effect through activation of an endogenous, gastric ghrelin system that is tightly coupled to \u03b2-adrenergic receptor signaling suggests the possibility of novel applications of H<sub>2<\/sub> therapy for various diseases.<\/p>\n<\/div>\n<div id=\"sec-a.i.f\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"sec-a.i.ftitle\">Methods<\/h2>\n<div id=\"sec-a.i.f.b\" class=\"sec sec-first\">\n<h3 id=\"sec-a.i.f.btitle\">Animals<\/h3>\n<p class=\"p p-first-last\">Procedures in animal experiments were approved by the university review board for animal care in Chiba University and Kyushu University, and performed in accordance with the guidelines established by the Science Council of Japan. All mice (C57BL\/6J) were maintained in the animal facility under controlled temperature and lighting (12-hour light, 12-hour dark), and received a standard mouse chow diet and filter-sterilized tap water <em>ad libitum<\/em>.<\/p>\n<\/div>\n<div id=\"sec-a.i.f.c\" class=\"sec\">\n<h3 id=\"sec-a.i.f.ctitle\">Preparation and administration of H<sub>2<\/sub>&#8211; water<\/h3>\n<p class=\"p p-first\">H<sub>2<\/sub> water was prepared by one of three methods. The first method utilized the spontaneous ionization reaction of magnesium in water<sup><a href=\"#b14\" rid=\"b14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a><\/sup>. A magnesium stick (AZ31, Nakagawa Metal, Japan, composed of 96% magnesium, 3% aluminum, and 1% zinc) was polished to remove the oxidized surface and wiped clean with 1\u2005N hydrogen chloride before dipping into drinking water for 15\u201320\u2005min at 25\u00b0C. The second method was based on the electrolysis of water with platinum electrodes at 150\u2005V DC for 20\u2005min (open-air water electrolysis method). The anode was placed inside a drinking straw to vent generated oxygen. In the third method (employed in the experiments with MPTP-induced PD model mice), H<sub>2<\/sub> gas was produced by solid polymer electrolyte water electrolysis method at 5\u2005V DC in which the cathode and anode were separated by a solid polymer electrolyte membrane (Nafion\u00ae E.I., du Pont de Nemours &amp; Co., Inc.) and collected through a polyethylene tube. Saturated H<sub>2<\/sub> water was then generated by bubbling water in a drinking bottle with H<sub>2<\/sub> gas such that H<sub>2<\/sub> gas filled the headspace. H<sub>2<\/sub> water made with this method and kept in sealed drinking bottles remained saturated with H<sub>2<\/sub> for at least 24\u2005hr.<\/p>\n<p class=\"p p-last\">In most experiments, each mouse received 0.8\u2005ml of H<sub>2<\/sub> water (administered within 30\u2005min of preparation) or control water (obtained by boiling H<sub>2<\/sub> water to degas) once every morning via feeding needle according to the experimental schedule. In experiments with MPTP-induced PD model mice, fresh H<sub>2<\/sub> water was supplied every 24\u2005hr and water intake was <em>ad libitum<\/em> for the 7 days prior to MPTP administration.<\/p>\n<\/div>\n<div id=\"sec-a.i.f.d\" class=\"sec\">\n<h3 id=\"sec-a.i.f.dtitle\">Measurements of ghrelin expression<\/h3>\n<p class=\"p p-first\">Male and female mice aged between 41 and 48 weeks were chosen for the initial screening for the altered expression of gastric enzymes (n = 4 for each group), as they are relatively stable in dietary intake, which may influence mostly on the digestive enzyme expressions. Since the previous study was done with male mice<sup><a href=\"#b14\" rid=\"b14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a><\/sup>, the following experiments chose male mice. For the time-course analysis of ghrelin induction by hydrogen water, male mice (16\u201321 weeks, average 17.9 weeks, n = 5\u201310 in each group) were selected. Adrenoceptor mediated changes of ghrelin secretion was analyzed in male mice (age between 11\u201313 weeks, n = 4\u20135 for each group). Of note, there was no body weight change more than one gram during the period of experiment (4 days). These results are in line with the previous report with a recombinant ghrelin injection to mice, where the body weight change was below one gram in three days<sup><a href=\"#b34\" rid=\"b34\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">34<\/a><\/sup>.<\/p>\n<p>Mice were administrated H<sub>2<\/sub> water or control water for 4 days according to the experimental schedule. At the end of the experiment, mice were sacrificed by cervical dislocation and blood was collected by cardiopuncture with EDTA as anti-coagulant. Blood was centrifuged at 2000\u2005g for 5\u2005min at 4\u00b0C and the plasma was collected and mixed with 1\u2005N hydrogen chloride (10% of plasma volume) to avoid inactivating deacylation of ghrelin<sup><a href=\"#b20\" rid=\"b20\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">20<\/a><\/sup>. Samples were stored at \u221280\u00b0C until analysis. Following blood collection, the stomach was removed intact and snap-frozen in liquid nitrogen for further analysis. For consistency, mice were killed 4\u20135\u2005hours after the final administration of H<sub>2<\/sub> water and at the same time of day. At the time of blood sampling the stomach was almost full of chow, which suggests that the water administration at a time (0.8\u2005ml\/time) through the feeding needle did not cause the sustained reduction on appetite.<\/p>\n<p class=\"p p-last\">The active form of ghrelin was measured in plasma by ELISA (Active Ghrelin ELISA kit, Sceti, Japan) according to the manufacturer&#8217;s instructions. For semi-quantitative PCR, stomach tissue frozen in liquid nitrogen was crushed into fine granules and total RNA was extracted with RNAiso (Takara, Japan) according to the manufacturer&#8217;s instructions. Two \u03bcg of total RNA was then used to synthesize first strand cDNA with a SuperScript VILO cDNA Synthesis Kit (Invitrogen, USA). mRNA expression levels were quantified by real-time PCR with SYBR green dye (Thunderbird Sybr qPCR Mix, Toyobo, Japan) with the specific primer sets shown in <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/table\/t1\/\" target=\"table\" class=\"fig-table-link figpopup\" rid-figpopup=\"t1\" rid-ob=\"ob-t1\" co-legend-rid=\"\" rel=\"noopener\"><span>Table 1<\/span><\/a>, and normalized to ribosomal protein L4 (RPL4) mRNA<sup><a href=\"#b35\" rid=\"b35\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">35<\/a><\/sup><sup>,<a href=\"#b36\" rid=\"b36\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">36<\/a><\/sup><sup>,<a href=\"#b37\" rid=\"b37\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">37<\/a><\/sup>.<\/p>\n<p><!--table ft1--><!--table-wrap mode=\"anchored\" t5--><\/p>\n<div class=\"table-wrap anchored whole_rhythm\" id=\"t1\">\n<h3>Table 1<\/h3>\n<p><!--caption a7--><\/p>\n<div class=\"caption\"><!--caption a8--><strong>Primer sets used for real-time PCR analyses<\/strong><\/div>\n<div class=\"xtable\">\n<table frame=\"hsides\" rules=\"groups\" border=\"1\" class=\"rendered small default_table\">\n<thead valign=\"bottom\">\n<tr>\n<th align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">RPL4.For<\/th>\n<th align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">GCCAAGACTATGCGCAGGAAT<\/th>\n<\/tr>\n<\/thead>\n<tbody valign=\"top\">\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">RPL4.Rev<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">GTAGCTGCTGCTTCCAGCTT<\/td>\n<\/tr>\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">Ghrelin.For<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">TCAAGCTGTCAGGAGCTCAGTA<\/td>\n<\/tr>\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">Ghrelin.Rev<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">TTGTCAGCTGGCGCCTCTT<\/td>\n<\/tr>\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">Somatostatin.For<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">TGGCTGCGCTCTGCATCGT<\/td>\n<\/tr>\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">Somatostatin.Rev<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">AGTACTTGGCCAGTTCCTGTT<\/td>\n<\/tr>\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">Gastrin.For<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">CAGCGCCAGTTCAACAAGCT<\/td>\n<\/tr>\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">Gastrin.Rev<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">ATTCGTGGCCTCTGCTTCTT<\/td>\n<\/tr>\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">b1AR.For<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">CACTGTGGACAGCGATTCGA<\/td>\n<\/tr>\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">b1AR.Rev<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">ACCTTGGACTCCGAGGAGAA<\/td>\n<\/tr>\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">b2AR.For<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">ACAGGAACTGCTGTGTGAGGAT<\/td>\n<\/tr>\n<tr>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">b2AR.Rev<\/td>\n<td align=\"justify\" valign=\"top\" charoff=\"50\" rowspan=\"1\" colspan=\"1\">ACGCTAAGGCTAGGCACAGT<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139809137747856\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4070541\/table\/t1\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<\/div>\n<\/div>\n<div id=\"sec-a.i.f.e\" class=\"sec\">\n<h3 id=\"sec-a.i.f.etitle\">Administration of ghrelin receptor antagonist, \u03b2<sub>1<\/sub>-adrenoceptor blocker, and MPTP<\/h3>\n<p class=\"p p-first-last\">The ghrelin receptor antagonist, D-Lys<sup>3<\/sup> GHRP-6 (Sigma-Aldrich, USA; 100\u2005nmol\/day), \u03b2<sub>1<\/sub>-adrenoceptor blocker, atenolol (ICI, USA; 10\u2005mg\/kg), or saline was administered by i.p. injection daily for 7 days in conjunction with supplying fresh control or H<sub>2<\/sub> water. On day 7, MPTP-HCl (Sigma-Aldrich, USA; 15\u2005mg\/kg in 0.9% NaCl per injection) or saline as control was administrated by i.p. injection four times at 2\u2005hr intervals. Mice were supplied with untreated tap water for the next 7 days before removal of brains under deep anesthesia (pentobarbital, 50\u2005mg\/kg i.p.).<\/p>\n<\/div>\n<div id=\"sec-a.i.f.f\" class=\"sec\">\n<h3 id=\"sec-a.i.f.ftitle\">Stereological and immunological analyses of nigral dopaminergic neurons<\/h3>\n<p class=\"p p-first\">Stereological analysis was carried out as described<sup><a href=\"#b14\" rid=\"b14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a><\/sup><sup>,<a href=\"#b38\" rid=\"b38\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">38<\/a><\/sup><sup>,<a href=\"#b39\" rid=\"b39\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">39<\/a><\/sup><sup>,<a href=\"#b40\" rid=\"b40\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">40<\/a><\/sup>. In brief, coronal sections (30\u2005\u03bcm thickness) were obtained through SNpc (\u22122.70\u2005mm to \u22123.80\u2005mm relative to bregma)<sup><a href=\"#b41\" rid=\"b41\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">41<\/a><\/sup> with a MICROM cryostat. Free-floating sections were incubated with Block Ace (Dainippon Pharmaceutics, Japan) for 30\u2005min followed by incubation with anti-tyrosine hydroxylase (TH) antibody (Chemicon, USA, 1:3000 in 10% Block Ace) for 2 days at 4\u00b0C. After rinsing, sections were immersed in a solution of 3% H<sub>2<\/sub>O<sub>2<\/sub> in methanol\/PBS (1:1) for 10\u2005min at room temperature, followed by incubation for 2\u2005hr in biotinylated goat anti-rabbit IgG (1:400, Vector, USA) and processing with a Vectastain ABC kit (Vector, USA) using 3\u20323\u2032-diaminobenzidinetetrahydrochloride (DAB, Vector, USA) as peroxidase chromogen.<\/p>\n<p>Unbiased stereological counts of TH-immunoreactive cell bodies in the SNpc were obtained using an optical fractionator method<sup><a href=\"#b42\" rid=\"b42\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">42<\/a><\/sup> and Stereo Investigator software (Stereo Investigator 8, MicroBrightField Inc., USA). The boundary of SNpc was delineated under 100 \u00d7 magnification and immunopositive neurons were counted in every third section (eight sections per brain) at 400 \u00d7 magnification on a Nikon ECLIPSE 80\u2005i microscope using a grid of 70 \u00d7 70\u2005\u03bcm on a counting grid (75 \u00d7 100 \u00d7 12\u2005\u03bcm) with 2\u2005\u03bcm upper and lower guard zones. Gundersen&#8217;s coefficient of error in all samples was &lt; 0.07.<\/p>\n<p class=\"p p-last\">Immunological detection and quantification of TH expression in the substantia nigra tissue were performed by western blotting with actin as control. Animals were prepared identically to those of the stereological analysis. The substantia nigra was removed and stored at \u221280\u00b0C until use. Samples were lysed on ice in hypotonic buffer (20\u2005mM Hepes pH 7.6, 10\u2005mM NaCl, 1.5\u2005mM MgCl<sub>2<\/sub>, 1\u2005mM EDTA, 0.1% Triton X-100) with protease inhibitor cocktail (Roche, USA), and cleared by a centrifugation at 20\u2005k G for 10\u2005min at 4\u00b0C. Following quantification of protein concentration using the BCA method with BSA as control, five \u03bcg of tissue lysate reduced in sample buffer was resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-TH antibody (1:2000) overnight at 4\u00b0C. Proteins were visualized using anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system (Immobilon Western Chemiluminescent HRP Substrate, Millipore, USA). Chemiluminescence image was quantified by Gauge application (Fuji Film, Japan) and the value was normalized to the level of actin band on the same sample specified by anti-Actin monoclonal antibody (Clone C4, Millipore, USA).<\/p>\n<\/div>\n<div id=\"sec-a.i.f.g\" class=\"sec sec-last\">\n<h3 id=\"sec-a.i.f.gtitle\">Statistical analysis and data managing<\/h3>\n<p class=\"p p-first-last\">The statistical significance of data was assessed by unpaired Student t-test (two-tailed) unless otherwise mentioned and results were considered significant at p \u2264 0.05.<\/p>\n<\/div>\n<\/div>\n<div id=\"sec-a.i.g\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"sec-a.i.gtitle\">Author Contributions<\/h2>\n<p class=\"p p-first-last\">A.M., Y.N., and M.N. conception and design of research; A.M., M.Y., T.T. performed experiments; A.M., M.Y., and M.N. analyzed data; A.M., M.Y., Y.N., and M.N. interpreted results of experiments; A.M. and M.Y. prepared figures; A.M., M.Y., Y.N., M.N., and H.N. wrote and revised manuscript; A.M., M.Y., T.T., Y.N., M.N., and H.N. approved final version of manuscript.<\/p>\n<\/div>\n<div id=\"ack-a.j.a\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"ack-a.j.atitle\">Acknowledgments<\/h2>\n<div class=\"sec\">\n<p>The authors are grateful to Dr. Douglas T. Hess (Case Western Reserve Univ.) for valuable advice and critical reading of the manuscript, and Dr. Yoshinori Tanaka (Corporate Engineering Division, Appliances Company, Panasonic Corporation, Japan) for help with hydrogen measurement. The authors also acknowledge the technical support of Mr. Yuichiro Kojima (Kyushu Univ.). This work was partly performed in the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University. This work is supported in part by Grant-in-Aid for Scientific Research on Innovative Areas (MEXT 20117008 to A.M.), Grant-in-Aid for Exploratory Research (JSPS 24659111 to A.M.), Grant-in-Aid for Scientific Research (B) (JSPS 23390053 to H.N.), Grant-in-Aid for Scientific Research (S) (JSPS 22221004 to Y.N.), Grant-in-Aid for Scientific Research (C) (JSPS 22590084 to M.N.) and Academic Challenge in Robert T. Huang Entrepreneurship of Kyushu University (to M.Y.).<\/p>\n<\/div>\n<\/div>\n<div id=\"ref-list-a.j.b\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"ref-list-a.j.btitle\">References<\/h2>\n<div class=\"ref-list-sec sec\" id=\"reference-list\">\n<ul class=\"back-ref-list\" style=\"list-style-type:decimal;\">\n<li id=\"b1\"><span class=\"mixed-citation\">Ohta S.<br \/>\n<span class=\"ref-title\">Recent Progress Toward Hydrogen Medicine: Potential of Molecular Hydrogen for Preventive and Therapeutic Applications<\/span>. <span class=\"ref-journal\">Curr. Pharm. 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[<a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/1793176\" ref=\"reftype=pubmed&amp;article-id=4070541&amp;issue-id=217774&amp;journal-id=1579&amp;FROM=Article%7CCitationRef&amp;TO=Entrez%7CPubMed%7CRecord\">PubMed<\/a>] <span class=\"nowrap\">[<a href=\"https:\/\/scholar.google.com\/scholar_lookup?journal=Anat.+Rec.&amp;title=Unbiased+stereological+estimation+of+the+total+number+of+neurons+in+thesubdivisions+of+the+rat+hippocampus+using+the+optical+fractionator&amp;author=M.+J.+West&amp;author=L.+Slomianka&amp;author=H.+J.+Gundersen&amp;volume=231&amp;publication_year=1991&amp;pages=482-97&amp;pmid=1793176&amp;\" target=\"_blank\" rel=\"noopener noreferrer\" ref=\"reftype=other&amp;article-id=4070541&amp;issue-id=217774&amp;journal-id=1579&amp;FROM=Article%7CCitationRef&amp;TO=Content%20Provider%7CLink%7CGoogle%20Scholar\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Google Scholar<\/a>]<\/span><\/span><\/li>\n<\/ul>\n<\/div>\n<\/div>\n<div style=\"display: none; width: 200px; top: -100px; left: -100px;\" aria-live=\"assertive\" aria-hidden=\"true\" class=\"ui-helper-reset ui-ncbipopper-wrapper ui-ncbilinksmenu\">\n<ul id=\"ui-ncbiinpagenav-2\">\n<li><a href=\"#abstract-a.h.b.ntitle\">Abstract<\/a><\/li>\n<li><a href=\"#sec-a.i.dtitle\">Results<\/a><\/li>\n<li><a href=\"#sec-a.i.etitle\">Discussion<\/a><\/li>\n<li><a href=\"#sec-a.i.ftitle\">Methods<\/a><\/li>\n<li><a href=\"#sec-a.i.gtitle\">Author Contributions<\/a><\/li>\n<li><a href=\"#ack-a.j.atitle\">Acknowledgments<\/a><\/li>\n<li><a href=\"#ref-list-a.j.btitle\">References<\/a><\/li>\n<\/ul>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Oral &#8216;hydrogen water&#8217; induces neuroprotective ghrelin secretion in mice<\/p>\n","protected":false},"author":1,"featured_media":17899,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[130],"tags":[],"disease":[890],"body-organ":[1019],"applications":[680],"test_subjects":[1518],"report-topic":[1435],"class_list":["post-27564","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-hydrogen-health","disease-parkinsons-disease-2","body-organ-brain-2","applications-ingestion-2","test_subjects-mouse-2","report-topic-ghrelin-secretion-2"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v28.1 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>H2O2 promotes ghrelin secretion for neuroprotection<\/title>\n<meta name=\"description\" content=\"Oral &#039;hydrogen water&#039; induces neuroprotective ghrelin secretion in mice\" \/>\n<meta name=\"robots\" content=\"index, follow, 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