Electrolyzed hydrogen water, method for producing same, method for activating intestinal barrier maintenance function using same, and method for regulating gene expression using same

Abstract

This electrolyzed hydrogen water has an activity of enhancing the intestinal barrier maintenance function by regulating expression of a response gene. The response gene is at least one kind of hub gene of CUL5 (Cullin5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p. This method for regulating gene expression by electrolyzed hydrogen water (excluding medical practice for humans) adjusts the dissolved hydrogen concentration to 1056-1080 ppb, and increases gene expression of CUL5 (Cullin5) and GOLGA7 (Golgin A7) and reduces gene expression of has-miR-429 and has-miR-200c-3p by the action of electrolyzed hydrogen water.

Description

Electrolyzed hydrogen water, its manufacturing method, and a method for activating intestinal barrier maintenance function and a method for regulating gene expression using the same

[0001] The present invention relates to electrolyzed hydrogen water, a method for producing the same, and a method for activating the intestinal barrier maintenance function and a method for regulating gene expression using the same.

[0002] The present applicant has been conducting various studies into new uses and functions of various hydrogen waters, such as electrolyzed hydrogen water, and mixtures containing such hydrogen waters. For example, Patent Document 1 proposes a hydrogen water mixture for suppressing alcoholic liver damage, which is a mixture of an ethanol solution and hydrogen water, with an ethanol concentration of 1-4% and a dissolved hydrogen concentration of 550-5600 ppb. This hydrogen water mixture is highly safe, easy to prepare, and inexpensive, and can effectively suppress alcoholic liver damage.

[0003] Japanese Patent Application Laid-Open No. 2022-126128

[0004] Incidentally, there are genes that are activated when cells are exposed to electrolyzed hydrogen water (hereinafter referred to as "response genes" that respond to electrolyzed hydrogen water). If the response genes can be identified, it is expected that the expression of the response genes can be regulated by electrolyzed hydrogen water. Furthermore, by regulating the expression of the response genes with electrolyzed hydrogen water, the biological function of the response genes can be imparted to the electrolyzed hydrogen water.

[0005] Therefore, the present invention has been made in consideration of these points, and aims to provide electrolyzed hydrogen water that has the activity of enhancing the biological function of response genes by regulating their expression, and a method for using the same.

[0006] As a result of intensive research to achieve the above-mentioned objective, the inventors identified four response genes that act as hub genes that respond to electrolyzed hydrogen water and that act to enhance the intestinal barrier maintenance function through immune regulation, and completed the electrolyzed hydrogen water of the present invention.

[0007] The electrolyzed hydrogen water of the present invention has the activity of enhancing the intestinal barrier maintenance function by regulating the expression of response genes, and is characterized in that the response genes are at least one hub gene selected from the group consisting of CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p.

[0008] The method for activating the intestinal barrier maintenance function of the present invention is a method for enhancing and activating the intestinal barrier maintenance function by regulating the expression of a response gene using electrolyzed hydrogen water, wherein the response gene is at least one hub gene selected from the group consisting of CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p, and the dissolved hydrogen concentration is adjusted to 1056 ppb or more and 1080 ppb or less.

[0009] The method for producing electrolyzed hydrogen water for activating intestinal barrier maintenance function of the present invention is a method for producing electrolyzed hydrogen water that enhances and activates intestinal barrier maintenance function by regulating the expression of a response gene, wherein the response gene is at least one hub gene selected from the group consisting of CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p, and the dissolved hydrogen concentration is adjusted to 1056 ppb or more and 1080 ppb or less.

[0010] The method for regulating gene expression using electrolyzed hydrogen water of the present invention (excluding medical procedures on humans) is a method for regulating gene expression using electrolyzed hydrogen water, characterized in that the gene responsive to the electrolyzed hydrogen water is at least one hub gene selected from the group consisting of CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p, the dissolved hydrogen concentration is adjusted to 1056 ppb or more and 1080 ppb or less, and the action of the electrolyzed hydrogen water increases gene expression of CUL5 (Cullin 5) and GOLGA7 (Golgin A7), while decreasing gene expression of has-miR-429 and has-miR-200c-3p.

[0011] According to the present invention, electrolyzed hydrogen water having the activity of enhancing the biological function of a response gene by regulating the expression of the response gene, and a method for using the same can be provided.

[0012] Figure 1 shows changes in gene expression in human colon cancer cells (Caco-2 cells) due to electrolyzed hydrogen water compared to purified water. Figure 2 shows the amount of change in gene expression in Caco-2 cells due to electrolyzed hydrogen water compared to purified water. Figure 3 shows the results of a Gene Ontology-based analysis of physiological function based on changes in gene expression in Caco-2 cells due to electrolyzed hydrogen water. Figure 4 shows the results of an analysis of transcription factors regulating gene expression based on changes in gene expression in Caco-2 cells due to electrolyzed hydrogen water. Figure 5 shows changes in microRNA (microRNA) expression in Caco-2 cells due to electrolyzed hydrogen water compared to purified water. Figure 6 shows the amount of change in microRNA expression in Caco-2 cells due to electrolyzed hydrogen water compared to purified water. Figure 7 shows the results of an analysis of the gene-microRNA network from the perspective of protein interactions for genes targeted by microRNAs whose expression is changed by electrolyzed hydrogen water in Caco-2 cells. FIG. 8 shows the results of an analysis of transcription factors common to hub genes that respond to electrolyzed hydrogen water in Caco-2 cells.

[0013] Based on the results of RNA sequencing (RNA-seq) analysis described in the Examples below, the inventors screened for differential expression of genes that respond to electrolyzed hydrogen water, and discovered the biological functions of the identified genes through gene ontology (GO) analysis and molecular interaction network analysis. The electrolyzed hydrogen water of the present invention was developed based on these findings.

[0014] The hub genes that respond to electrolyzed hydrogen water are CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p (hereinafter collectively referred to as "specific response genes"). In the electrolyzed hydrogen water of the present invention, the response gene is specified to be at least one hub gene selected from the group consisting of CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p (hereinafter also referred to as "response gene-specific electrolyzed hydrogen water").

[0015] Gene expression of CUL5 (Cullin 5) and GOLGA7 (Golgin A7) is increased by the action of electrolyzed hydrogen water. On the other hand, gene expression of has-miR-429 and has-miR-200c-3p is decreased by the action of electrolyzed hydrogen water. In other words, the expression levels of these specific response genes can be regulated using electrolyzed hydrogen water.

[0016] The transcription factor KLF5 targets CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p. Specific response genes in intestinal epithelial-like cells treated with electrolyzed hydrogen water are thought to be regulated by KLF5.

[0017] Furthermore, the regulation of expression of specific response genes by electrolyzed hydrogen water acts to enhance the intestinal barrier maintenance function through immune control, which is the biological function of specific response genes. In other words, response gene-specific electrolyzed hydrogen water can be said to have the activity of enhancing the intestinal barrier maintenance function by regulating the increase or decrease in expression of specific response genes.

[0018] The influence of specific response genes on intestinal function is described in, for example, the following paper: 1) Yu, T., et al., Overexpression of miR-429 impairs intestinal barrier function in diabetic mice by down-regulating occludin expression. Cell Tissue Res, 2016. 366(2): p. 341-352. 2) He, S., et al., Ferulic Acid Ameliorates Lipopolysaccharide-Induced Barrier Dysfunction via MicroRNA-200c-3p-Mediated Activation of PI3K / AKT Pathway in Caco-2 Cells. Front Pharmacol, 2020. 11: p. 376. 3) Mo, JS, et al., MicroRNA 429 Regulates Mucin Gene Expression and Secretion in Murine Model of Colitis. J Crohns Colitis, 2016. 10(7): p. 837-49. 4) Chen, J., et al., The roles of miR-200c in colon cancer and associated molecular mechanisms. Tumour Biol, 2014. 35(7): p. 6475-83.

[0019] Furthermore, response gene-specific electrolyzed hydrogen water is expected to function in controlling the activation or inhibition of autophagy and preventing the progression of cancer by enhancing the intestinal barrier maintenance function.

[0020] Autophagy is an intracellular recycling system (a degradation system using lysosomes) that breaks down intracellular components, purifying them and removing harmful substances, thereby preventing various diseases such as cancer. In addition to its role as a mechanism for the breakdown of intracellular components, autophagy is also known to function in regulating biological mechanisms through the secretion of intracellular components. In particular, secretory autophagy (exophagy, the extracellular release of secreted products) has been shown to be involved in the regulation of inflammation and the exchange of metabolites, which are associated with various diseases.

[0021] It has been reported that the miR-200c family may affect cancer progression. Overexpression of miR-200c promotes cancer proliferation, migration, and invasion. On the other hand, decreased expression of miR-200c has been demonstrated to suppress cancer proliferation, migration, and invasion. Therefore, response gene-specific electrolyzed hydrogen water not only acts to enhance intestinal barrier maintenance function through immune regulation by suppressing the expression of the specific response gene hsa-miR-200c-3p, but may also function in cancer prevention.

[0022] It has also been reported that decreased expression of hsa-miR-200c-3p promotes cell adhesion to fibronectin, a part of the extracellular matrix that forms the scaffold. Therefore, it is possible that response gene-specific electrolyzed hydrogen water maintains the formation of cell-cell adhesion by suppressing the expression of the specific response gene hsa-miR-200c-3p.

[0023] The miR-429 family is a member of the miR-200 family and is known to have similar functions. Therefore, it is thought that response gene-specific electrolyzed hydrogen water functions to maintain intestinal barrier function through suppression of the expression of the specific response gene has-miR-429. In vivo studies in mice have reported that miR-429 impairs intestinal barrier function by reducing the expression of genes involved in barrier function.

[0024] Increased expression of miR-200c or miR-429 has been shown to promote cellular inflammatory responses in diabetic mouse models.

[0025] Response gene-specific electrolyzed hydrogen water can be produced using a commercially available hydrogen generator (such as an electrolyzed water generator). Therefore, response gene-specific electrolyzed hydrogen water can be said to be highly safe, easy to prepare using commercially available equipment, and inexpensive.

[0026] From the viewpoint of regulating the expression of specific response genes, the dissolved hydrogen concentration in the response gene-specific electrolyzed hydrogen water is preferably 550 ppb or more and 5600 ppb or less, more preferably 800 ppb or more and 1320 ppb or less, and even more preferably 1056 ppb or more and 1080 ppb or less.

[0027] The response gene-specific electrolyzed hydrogen water constructed as described above has the activity of enhancing the intestinal barrier maintenance function, which is the biological function of these hub genes, by regulating the expression of at least one hub gene selected from the group consisting of CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p, which have been identified as response genes.

[0028] The present invention will be described below based on examples. However, the present invention is not limited to these examples, and these examples can be modified or changed based on the spirit of the present invention, and such modifications are not excluded from the scope of the present invention.

[0029] <Production of electrolyzed hydrogen water> Using an electrolyzed water generator (manufactured by Nippon Trim Co., Ltd., product name: TRIMION GRACE), electrolyzed hydrogen water with a dissolved hydrogen concentration of Level 4 was produced at a temperature of 22°C and a flow rate of 1.5 L / min, and purified water was also obtained by filtering using microcarbon.

[0030] Next, the pH and dissolved hydrogen concentration of the electrolyzed hydrogen water at level 4 were measured. The pH was measured using a pH meter (manufactured by HORIBA, product name: LAQUA act D-71), and the dissolved hydrogen concentration was measured using a dissolved hydrogen meter DH-35A (manufactured by Toa DKK Corporation). Level 4: 1320 ppb (up to 1350 ppb), pH 10.

[0031] <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> 5x Dulbecco's modified Eagle's medium (hereinafter referred to as "DMEM", Wako, 044-29765) prepared from powder was diluted 5 times with the above-mentioned purified water or level 4 electrolyzed hydrogen water, and used as a medium for treating electrolyzed hydrogen water (i.e., a treatment medium that is a hydrogen water mixture made by mixing 20% ​​5x DMEM and 80% electrolyzed hydrogen water or purified water). The dissolved hydrogen concentration in level 4 electrolyzed hydrogen water medium is as follows: Level 4: 1320 ppb (up to 1350 ppb) x 4 / 5 = 1056 ppb (up to 1080 ppb).

[0032] <Cell culture> Caco2 cells (RIKEN BRC), a human colon carcinoma cell line, were cultured in electrolyzed hydrogen water medium containing 10% fetal bovine serum (FBS; Sigma-Aldrich, F7524) and 1% penicillin-streptomycin (Wako, 168-23191) at 37°C and 5% CO 2 It was cultivated in an atmosphere.

[0033] Transcriptome Sequencing: Samples extracted from six independent wells were used for RNA-seq analysis. A cDNA library was constructed using the NEB Next® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs). The final cDNA library was prepared through several rounds of purification, end repair, A-tailing, sequence adapter ligation, size selection, and PCR enrichment. RNA sequencing was performed on the library using a Novogene NovaSeq 6000 System. Reads were mapped to reference sequences using TopHat 2. Three samples were randomly selected from the ACW and EHW-exposure groups for transcriptome sequencing. TRIzol™ Reagent (Invitrogen) was used to extract total RNA from the thymus specimens. Total RNA was sequenced using the NEB Next Multiplex Directional RNA Library Prep Kit and the NEB Small RNA Library Kit. After quantification and identification, the total RNA was used as input material for the synthesis of mRNA and microRNA libraries. All transcriptome sequencing analyses were performed on the Illumina HiSeq™ 2500 platform.

[0034] <Differentially expressed gene analysis> Differentially expressed genes were screened using the EBSeq toolkit, and genes with differential expression levels of FDR < 0.05 were identified using the Benjamin-Hochberg test. The results are shown in Figures 1 to 4. The results for microRNAs are shown in Figures 5 and 6.

[0035] Figure 1 is a volcano plot showing the change in gene (mRNA) expression in human colon cancer cells (Caco-2 cells) treated with electrolyzed hydrogen water (EHW) compared with purified water (ACW). The vertical axis of Figure 1 indicates the statistical significance level, with higher values ​​indicating greater statistical significance. The horizontal axis of Figure 1 indicates gene (mRNA) expression levels in response to electrolyzed hydrogen water. Values ​​less than 0 indicate genes with decreased expression levels, while values ​​greater than 0 indicate genes with increased expression levels. From Figure 1, 82 differentially expressed genes (DEGs) were extracted between purified water and electrolyzed hydrogen water (false discovery rate: FDR: p < 0.05). Of the 82, 44 were differentially expressed genes (e.g., ARNTL2, MACC1, LIN7C, EEA1, ARL5B, STARD4) that showed increased gene expression (increased mRNA levels). On the other hand, 38 genes were differentially expressed (RPS28, MT-ND5, RPL13, RPL31, C21orf33, etc.) that showed decreased gene expression (decreased mRNA levels).

[0036] Figure 2 is a heatmap showing the amount of change (degree of change) in the expression of genes in human colon cancer cells (Caco-2 cells) that was altered by electrolyzed hydrogen water (EHW) compared to purified water (ACW) using color shading. The heatmap was created by standardizing and comparing data from two independent experiments. The letters in Figure 2 represent the gene names. In Figure 2, the upper part shows genes whose expression increased with electrolyzed hydrogen water (MANEA to PUS7L), and the lower part shows genes whose expression decreased with electrolyzed hydrogen water (MT-ND5 to GALK1).

[0037] Figure 3 shows a Gene Ontology (GO)-based biological function analysis of human colon cancer cells (Caco-2 cells). Specifically, Figure 3 shows the results of a GO-based physiological function analysis (shown from top to bottom in Figure 3 as molecular functions, biological processes, and cellular components) based on changes in gene expression due to EHW. GO analysis was performed using the clusterProfiler package, a statistical analysis software application written in the R language. The top 10 GO terms with the lowest p-values ​​(p < 0.05) were selected and visualized. Figure 3 shows that among the genes whose expression was altered by EHW, GO terms (functional terms) related to protein synthesis, transport, and cell-cell adhesion were highly profiled. Specifically, GO terms for protein synthesis include, for example, rRNA binding, large ribosomal subunit rRNA binding, translation, ribosome assembly, peptide biosynthetic process, cytoplasmic translation, cellular macromolecule biosynthetic process, small ribosomal subunit, ribosome, polysomal ribosome, large ribosomal subunit, and cytosolic small ribosomal subunit. GO terms for transport include, for example, proton transmembrane transporter activity, oxidation reduction-driven active transmembrane transporter activity, and active ion transmembrane transporter activity. GO terms for cell-cell adhesion include, for example, focal adhesion and cell-substrate junction.

[0038] Figure 4 shows the analysis of transcription factors controlling differentially expressed genes (DEGs) in human colon cancer cells (Caco-2 cells) (enrichment analysis, ChIP-X Enrichment Analysis 3: ChEA3). Specifically, Figure 4 shows the results of analyzing transcription factors controlling gene expression based on changes in gene expression caused by electrolyzed hydrogen water (EHW). ChIP-X Enrichment Analysis 3 (ChEA3) was used for the transcription factor enrichment analysis. In the ChEA3 analysis, the top 10 factors with the lowest p-values ​​(p < 0.05) were selected and visualized. Figure 4 shows that Gut-Enriched Krueppel-Like Factor (KLF4), a transcription factor associated with enhanced skin barrier function (intestinal barrier function), was enriched as a transcription factor targeting genes with differentially expressed DEGs in EHW. KLF4 has also been suggested to function as a colon cancer suppressor.

[0039] Figure 5 is a volcano plot showing the change in microRNA expression in human colon cancer cells (Caco-2 cells) treated with electrolyzed hydrogen water (EHW) compared to purified water (ACW). As with Figure 1, the vertical axis of Figure 5 indicates the statistical significance level, with higher values ​​indicating greater statistical significance. The horizontal axis of Figure 5 indicates the expression level of microRNAs treated with electrolyzed hydrogen water. Values ​​less than 0 indicate microRNAs with decreased expression levels, while values ​​greater than 0 indicate microRNAs with increased expression levels. From Figure 5, 50 differentially expressed genes (DEGs) were extracted between purified water and electrolyzed hydrogen water (extracted with FDR p < 0.05). Of the 50 genes, 27 are differentially expressed genes that show increased microRNA expression (e.g., hsa-miR-148a-3p, hsa-miR-215-5p, hsa-miR-192-5p, hsa-miR-27b-3p, hsa-miR-3529-3p, hsa-miR-206), while 23 are differentially expressed genes that show decreased microRNA expression (e.g., hsa-miR-373-3p, hsa-miR-372-3p, hsa-miR-30d-5p, hsa-miR-200c-3p, hsa-miR-7-5p, hsa-miR-125a-5p, hsa-miR-7-5p, hsa-miR-516b-5p).

[0040] Figure 6 is a heatmap showing the amount of variation (degree of variation) in the expression of microRNAs in human colon cancer cells (Caco-2 cells) that was altered by electrolyzed hydrogen water (EHW) compared to purified water (ACW) using color shading. The heatmap was created by standardizing and comparing data from two independent experiments. The letters in Figure 5 represent gene names. In Figure 5, the upper panel shows microRNAs whose expression was increased by electrolyzed hydrogen water (hsa-miR-192-5p to hsa-miR-200a-5p), and the lower panel shows microRNAs whose expression was decreased by electrolyzed hydrogen water (hsa-miR-194-5p to hsa-miR-182-5p).

[0041] <Gene prediction and construction of interaction network> MicroRNA targets were predicted from differentially expressed genes using TargetScan (https: / / www.targetscan.org / vert_72 / ), and an mRNA-microRNA association network was constructed. TargetScan is a microRNA target prediction algorithm and its database. MCODE was also used to identify hub genes within the interaction network. MCODE is a method for discovering protein complexes from protein-protein interaction (PPI) networks. The results are shown in Figure 7.

[0042] Figure 7 shows the mRNA-microRNA interaction analysis using TargetScan and MCODE in human colon cancer cells (Caco-2 cells). Specifically, Figure 4 shows the results of using TargetScan to screen mRNAs targeted by microRNAs affected by electrolyzed hydrogen water (EHW) from genes affected by EHW, followed by analysis of the protein-protein interaction (PPI) network using MCODE. Figure 7 shows the hub genes selected as CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p. CUL5 and GOLGA7 are upregulated genes showing increased expression. Meanwhile, has-miR-429 and has-miR-200c-3p are downregulated genes showing decreased expression.

[0043] <Prediction and selection of hub genes regulating transcription factors (TFs)> When detecting mRNA-microRNA interactions, TFs were considered as genes. TF-mRNA and TF-microRNA pairs were obtained using the following three transcription factor databases: hTFtarget (https: / / ngdc.cncb.ac.cn / databasecommons / database / id / 6946), motifmap (http: / / motifmap.ics.uci.edu / ), and TransmiR v2.0 (http: / / www.cuilab.cn / transmir). The results are shown in Figure 8.

[0044] Figure 8 shows the results of an analysis of transcription factors common to the hub genes responsive to electrolyzed hydrogen water (EHW) in human colon cancer cells (Caco-2 cells) using the three transcription factor databases. Specifically, Figure 8 shows the results of an analysis of transcription factors common to the four hub genes identified above (CUL5, GOLGA7, has-miR-429, and has-miR-200c-3p). Figure 8 shows that by comparing the three transcription factor databases, the 16 transcription factors listed in Table 1 below were identified as common transcription factors regulating the four hub genes: CUL5, GOLGA7, has-miR-429, and has-miR-200c-3p. Among transcription factors, KLF4 (Krueppel-Like Factor 4) in particular is thought to be highly likely to be involved in the regulation of CUL5, GOLGA7, has-miR-429 and / or has-miR-200c-3p in the intestinal epithelium.

[0045]

[0046] <Summary> The above analysis identified four responsive genes, CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p, as hub genes that respond to electrolyzed hydrogen water with a dissolved hydrogen concentration of 1056 ppb or more and 1080 ppb or less. By regulating the increase or decrease in expression of these responsive genes, the electrolyzed hydrogen water of the present invention acts to enhance the intestinal barrier maintenance function through immune control, i.e., it has the activity of enhancing the intestinal barrier maintenance function.

[0047] An example of the application of the present invention is electrolyzed hydrogen water, which has the activity of enhancing the intestinal barrier maintenance function.

Claims

1. Electrolyzed hydrogen water having the activity of enhancing intestinal barrier maintenance function by regulating the expression of a response gene, wherein the response gene is at least one hub gene selected from the group consisting of CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p.

2. Electrolyzed hydrogen water according to claim 1, characterized in that the dissolved hydrogen concentration is 1056 ppb or more and 1080 ppb or less.

3. A method for enhancing and activating intestinal barrier maintenance function by regulating the expression of a response gene using electrolyzed hydrogen water, wherein the response gene is at least one hub gene selected from the group consisting of CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429 and has-miR-200c-3p, and the dissolved hydrogen concentration is adjusted to 1056 ppb or more and 1080 ppb or less.

4. A method for producing electrolyzed hydrogen water that enhances and activates intestinal barrier maintenance function by regulating the expression of a response gene, wherein the response gene is at least one hub gene selected from the group consisting of CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429 and has-miR-200c-3p, and the dissolved hydrogen concentration is adjusted to 1056 ppb or more and 1080 ppb or less.

5. A method for regulating gene expression using electrolyzed hydrogen water, wherein the gene responsive to the electrolyzed hydrogen water is at least one hub gene selected from the group consisting of CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429 and has-miR-200c-3p, the dissolved hydrogen concentration is adjusted to 1056 ppb or more and 1080 ppb or less, and the gene expression of CUL5 (Cullin 5) and GOLGA7 (Golgin A7) is increased while the gene expression of has-miR-429 and has-miR-200c-3p is decreased by the action of the electrolyzed hydrogen water (however, medical procedures on humans are excluded).

Images