Gene expression profiling in rat liver after H2 administration with or without dietary stress

Molecular hydrogen (H2) is a biologically active gas that is used medically to ameliorate various systemic pathological conditions. H2 also regulates gene expression involved in intracellular signaling and metabolic pathways. Therefore, we attempted to identify genes that exhibit similar changes in expression in response to H2 by employing DNA microarrays and gene set enrichment analysis. We found that H2 activated the expression of sets of genes regulated by histone H3K27 methylation status. H2 also modified the expression of many genes regulated by a wide variety of signaling pathways. We divided 4-week-old male F344 rats into four groups: 1) control/normal diet group: fed with basal rodent chow (CE-2; CLEA Japan Inc., Tokyo, Japan) and reared in air (n = 4), 2) hydrogen-treated/normal diet group: fed with basal rodent chow and hydrogen-rich water in 2% hydrogen/98% air (n = 4), 3) control/CDAA diet group: fed with a choline-deficient L-amino acid-defined diet (CDAA diet; A06083101, Research Diets, Inc., New Brunswick, NJ) and reared in air (n = 4), 4) hydrogen-treated/CDAA diet group: fed with CDAA diet and hydrogen-rich water in 2% hydrogen/98% air (n = 4). Hydrogen-rich water (0.7mM dissolved hydrogen) was generated from distilled water with 0.44 mM Na2SO4 using Aquela Blue, a water-electrolyzing device to produce electrolyzed hydrogen-saturated water near neutral pH (MiZ Co., Ltd, Fujisawa, Japan). The control dehydrogenized water was prepared by gently stirring hydrogen-rich water in open air for 24 hours. Hydrogen-rich water or control water was administrated ad libitum to the rats with a 50-ml closed glass vessel equipped with an outlet line having a ball bearing. After 3-week rearing, the rats were sacrificed and their livers were excised for RNA extraction. Furthermore, we performed DNA microarray analysis to examine changes in the gene expression profiles.

分子氢（Molecular hydrogen，H₂）是一种具有生物活性的气体，临床可用于改善多种全身性病理状态。此外，H₂还可调控参与细胞内信号转导与代谢通路的基因表达。因此，本研究借助DNA微阵列（DNA microarray）与基因集富集分析（gene set enrichment analysis），旨在筛选出对H₂处理呈现相似表达变化的基因。研究发现，H₂可激活受组蛋白H3K27甲基化（histone H3K27 methylation）状态调控的基因集的表达；同时，H₂还可改变大量受多种信号通路调控的基因的表达水平。

本研究将4周龄雄性F344大鼠分为四组：
1) 正常饮食对照组：饲喂基础啮齿类饲料（CE-2；日本东京CLEA日本公司），于正常空气环境中饲养（n=4）；
2) 正常饮食氢处理组：饲喂基础啮齿类饲料，于2%氢气与98%空气的混合环境中饲养并自由饮用富氢水（n=4）；
3) CDAA饮食对照组：饲喂胆碱缺乏型L-氨基酸限定饲料（choline-deficient L-amino acid-defined diet，CDAA diet；货号A06083101，美国新泽西州Research Diets公司），于正常空气环境中饲养（n=4）；
4) CDAA饮食氢处理组：饲喂CDAA饲料，于2%氢气与98%空气的混合环境中饲养并自由饮用富氢水（n=4）。

富氢水（溶解氢浓度为0.7mM）以含0.44mM硫酸钠的蒸馏水为原料，通过电解装置Aquela Blue制备，该装置可生成接近中性pH的电解氢饱和水（日本藤泽市MiZ株式会社）。对照脱氢水的制备方法为：将富氢水置于敞口环境中轻柔搅拌24小时。

本研究采用配备带滚珠轴承出口管路的50ml密闭玻璃容器，为大鼠自由提供富氢水或对照水。饲养3周后，处死大鼠并摘取肝脏用于RNA提取。此外，本研究通过DNA微阵列分析检测基因表达谱的变化情况。