Electrolyzed hydrogen water

Abstract

To provide electrolyzed hydrogen water having an activity of enhancing the biological function of a response gene by regulating expression of the response gene.SOLUTION: Electrolyzed hydrogen water has an activity of enhancing the intestinal barrier maintenance function by regulating 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.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to electrolyzed hydrogen water.

Background Art

[0002] The applicant of the present application has been variously studying new uses and functions of various hydrogen waters such as electrolyzed hydrogen water and mixtures containing the same. For example, in Patent Document 1, a hydrogen water mixture for suppressing alcoholic liver injury is proposed in which an ethanol solution and hydrogen water are mixed, the ethanol concentration is 1 to 4%, and the dissolved hydrogen concentration is 550 to 5600 ppb. This hydrogen water mixture is highly safe, easy and inexpensive to prepare, and can effectively suppress alcoholic liver injury.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] By the way, there are genes that are activated when electrolyzed hydrogen water acts on cells (hereinafter, also referred to as "response genes" that respond to electrolyzed hydrogen water). If the response genes can be identified, it is expected to regulate the expression of the response genes with electrolyzed hydrogen water. And by regulating the expression of the response genes by electrolyzed hydrogen water, the biological functions of the response genes can be imparted to electrolyzed hydrogen water.

[0005] Therefore, the present invention has been made in view of such points, and an object thereof is to provide electrolyzed hydrogen water having an activity of enhancing the biological function of a response gene by regulating the expression of the response gene.

Means for Solving the Problems

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

[0007] The electrolyzed hydrogen water of the present invention has an activity of enhancing the intestinal barrier maintenance function by regulating the expression of response genes, and is characterized in that 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.

Effect of the Invention

[0008] According to the present invention, it is possible to provide electrolyzed hydrogen water having an activity of enhancing the biological function of a response gene by regulating the expression of the response gene.

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] Based on the analysis results of RNA sequencing (RNA-seq) described in the following examples, the present inventors screened for differential expression of genes responsive to electrolyzed hydrogen water, and found the biological functions of the genes identified therein by gene ontology (GO) analysis and molecular interaction network analysis. The electrolyzed hydrogen water of the present invention was completed based on these findings.

[0011] The hub genes responsive to electrolyzed hydrogen water are CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p (hereinafter collectively also referred to as "specific response genes"). In the electrolyzed hydrogen water of the present invention, it is specified that 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 (hereinafter also referred to as "response gene - specified electrolyzed hydrogen water").

[0012] CUL5 (Cullin 5) and GOLGA7 (Golgin A7) have increased gene expression due to the action of electrolyzed hydrogen water. On the other hand, has-miR-429 and has-miR-200c-3p have decreased gene expression due to the action of electrolyzed hydrogen water. That is, these specific response genes can be used to regulate their expression levels using electrolyzed hydrogen water.

[0013] In addition, the transcription factor KLF5 targets CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p. The specific response genes in intestinal epithelial-like cells of electrolyzed hydrogen water for response gene identification are considered to be under the control of KLF5.

[0014] And the regulation of the expression of specific response genes by electrolyzed hydrogen water acts on enhancing the intestinal barrier maintenance function by immune regulation, which is the biological function of specific response genes. In other words, it can be said that electrolyzed hydrogen water for response gene identification has the activity to enhance the intestinal barrier maintenance function by regulating the up-regulation or down-regulation of the expression of specific response genes.

[0015] In addition, the fact that specific response genes affect intestinal function is described in, for example, the following papers. 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, J.S., 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.。

[0016] In addition, in the response gene-specific electrolyzed hydrogen water, it is expected to function in controlling the activation or suppression of autophagy and preventing cancer progression by enhancing the intestinal barrier maintenance function.

[0017] Autophagy is an intracellular recycling system (a degradation system by lysosomes), which purifies intracellular components and removes harmful substances by degrading intracellular components, and works in preventing various diseases such as cancer. In addition to its role as a degradation mechanism of intracellular components, autophagy is also known to function in regulating biological mechanisms by secreting intracellular components. In particular, secretory autophagy (exophagy, extracellular release of secretions) has been shown to be involved in the regulation of inflammation related to various diseases and the exchange of metabolites.

[0018] The miR-200c family has been reported to potentially affect cancer progression. Overexpression of miR-200c promotes cancer cell proliferation, migration, invasion, etc. On the other hand, it has been demonstrated that a decrease in miR-200c expression suppresses cancer cell proliferation, migration, invasion, etc. Therefore, in the response gene-specific electrolyzed hydrogen water, it may not only act on enhancing the intestinal barrier maintenance function by immune regulation through suppressing the expression of the specific response gene hsa-miR-200c-3p, but also function in cancer prevention.

[0019] Furthermore, it has been reported that a decrease in the expression of hsa-miR-200c-3p promotes cell adhesion to fibronectin, which is one of the extracellular matrices that form the scaffold. Therefore, in the response gene-specific electrolyzed hydrogen water, it may be possible to maintain the formation of cell-cell adhesion by suppressing the expression of the specific response gene hsa-miR-200c-3p.

[0020] The miR-429 family is a member of the miR-200 family and is known to have similar functions. Therefore, in the response gene-specific electrolyzed hydrogen water, it is considered to function in maintaining the intestinal barrier function by suppressing the expression of the specific response gene has-miR-429. In in vivo studies of mice, it has been reported that miR-429 impairs the intestinal barrier function by decreasing the expression of genes involved in the barrier function.

[0021] An increase in the expression of miR-200c or miR-429 has been found to promote the inflammatory response of cells in a diabetic mouse model.

[0022] The response gene-specific electrolyzed hydrogen water can generally be produced using commercially available hydrogen generation devices (such as electrolyzed water generation devices). Therefore, it can be said that the response gene-specific electrolyzed hydrogen water is highly safe, easy to prepare with a commercially available device, and inexpensive.

[0023] From the perspective 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.

[0024] The response gene-specific electrolyzed hydrogen water configured 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 are specified as response genes.

Example

[0025] The present invention will be described below based on examples. It should be noted that the present invention is not limited to these examples, and these examples can be modified and changed based on the gist of the present invention, and they are not excluded from the scope of the present invention.

[0026] <Production of electrolyzed hydrogen water> Using an electrolyzed water generator (manufactured by Nippon Trim Co., Ltd., product name: TRIMION GRACE), under the conditions of a temperature of 22°C and a flow rate of 1.5 L / min, electrolyzed hydrogen water with a dissolved hydrogen concentration level of 4 was obtained, and purified water filtered by microcarbon was also obtained.

[0027] Next, the pH and dissolved hydrogen concentration of the electrolyzed hydrogen water at level 4 were measured. For the measurement of pH, a pH meter (manufactured by HORIBA, product name: LAQUA act D-71) was used, and for the measurement of the dissolved hydrogen concentration, a dissolved hydrogen meter DH-35A (manufactured by Toa DKK Corporation) was used. · Level 4: 1320 ppb (~1350 ppb), pH 10.

[0028] <Preparation of medium for treating electrolyzed hydrogen water (electrolyzed hydrogen water medium)> 5× Dulbecco's modified Eagle's medium (hereinafter referred to as "DMEM", Wako, 044-29765) prepared from powder was diluted 5-fold with the above-mentioned purified water or electrolyzed hydrogen water at level 4, and used as a medium for treating electrolyzed hydrogen water (that is, a hydrogen water mixture obtained by mixing 20% of 5× DMEM and 80% of electrolyzed hydrogen water or purified water). The dissolved hydrogen concentration in the electrolyzed hydrogen water medium at level 4 is as follows. · Level 4: 1320 ppb (~1350 ppb) × 4 / 5 = 1056 ppb (~1080 ppb).

[0029] <Cell culture> The Caco2 cells (RIKEN BRC), a human colorectal cancer cell line, were cultured at 37 °C in an electrolytic hydrogen water medium containing 10% fetal bovine serum (FBS; Sigma-Aldrich, F7524) and 1% penicillin-streptomycin (Wako, 168-23191) in a 5% CO 2 atmosphere.

[0030] <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). Purification, end repair, A-tailing, sequence adapter ligation, size selection, and PCR enrichment were repeated several times to prepare the final cDNA library. RNA sequencing was performed on the library using the NovaSeq 6000 System from Novogene. Reads were mapped to the reference sequence using TopHat 2. For transcriptome sequencing, three samples were randomly selected from the ACW group and the EHW-exposure group. TRIzol® reagent (Invitrogen) was used to extract total RNA from 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, total RNA was used as input material for the synthesis of mRNA and microRNA libraries. All transcriptome sequence analyses were performed on the Illumina HiSeq™ 2500 platform.

[0031] <Differential Gene Expression Analysis> Differentially expressed genes were screened using the EBSeq toolkit, and genes with expression variation were identified with FDR < 0.05 by the Benjamin-Hochberg test. The results are shown in Figures 1 to 4. In addition, the results for microRNA are shown in Figures 5 and 6.

[0032] Figure 1 is a Volcano plot showing the expression variation of genes (mRNA) by electrolyzed hydrogen water (EHW) with respect to purified water (ACW) in human colorectal cancer cells (Caco-2 cells). The vertical axis in Figure 1 indicates the statistical significance level, and the higher the numerical value, the more statistically significant it is. The horizontal axis in Figure 1 indicates the expression level of genes (mRNA) by electrolyzed hydrogen water. Values smaller than 0 indicate genes with decreased expression levels, while values larger than 0 indicate genes with increased expression levels. From Figure 1, 82 differentially expressed genes (DEG) were extracted between purified water and electrolyzed hydrogen water (extracted at p < 0.05 in the False Discovery Rate: FDR). Among the 82 genes, 44 are differentially expressed genes showing an increase in gene expression (increase at the mRNA level) (such as ARNTL2, MACC1, LIN7C, EEA1, ARL5B, STARD4, etc.). On the other hand, 38 are differentially expressed genes showing a decrease in gene expression (decrease at the mRNA level) (such as RPS28, MT-ND5, RPL13, RPL31, C21orf33, etc.).

[0033] Figure 2 is a Heatmap showing the amount of variation (degree of variation) with respect to purified water (ACW) for genes whose expression was varied by electrolyzed hydrogen water (EHW) in human colorectal cancer cells (Caco-2 cells), indicated by the shade of color, etc. The Heatmap compares the standardized experimental data for two sets that are independent of each other. Note that the English letters in Figure 2 represent gene names. In Figure 2, the upper side shows genes whose expression increased with electrolyzed hydrogen water (MANEA~PUS7L), and the lower side shows genes whose expression decreased with electrolyzed hydrogen water (MT-ND5~GALK1).

[0034] Figure 3 shows the biological function analysis based on Gene Ontology (GO) in human colorectal cancer cells (Caco-2 cells). Specifically, Figure 3 shows the results of analyzing physiological functions (in the order from the top of Figure 3: Molecular Functions, Biological Processes, and Cellular Components) based on GO from gene expression fluctuations caused by electrolyzed hydrogen water (EHW). For the GO analysis, the clusterProfiler package, which is an application of statistical analysis software using the R language, was used. In the GO analysis, the top 10 with the smallest p-value at p < 0.05 were selected and visualized. From Figure 3, among the genes with expression fluctuations observed with electrolyzed hydrogen water, GO Terms (function terms) related to protein synthesis, transport, and cell-cell adhesion were profiled at the top. Specifically, examples of GO Terms for protein synthesis include 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, cytosolic small ribosomal subunit, etc. Examples of GO Terms for transport include proton transmembrane transporter activity, oxidoreduction-driven active transmembrane transporter activity, active ion transmembrane transporter activity, etc. Examples of GO Terms for cell-cell adhesion include focal adhesion, cell-substrate junction, etc.

[0035] Figure 4 shows the analysis of transcription factors that regulate differentially expressed genes (DEGs) in human colorectal cancer cells (Caco-2 cells) (enrichment analysis, ChIP-X Enrichment Analysis 3: ChEA3). Specifically, Figure 4 shows the results of analyzing transcription factors that control the expression of genes from gene expression fluctuations caused by electrolyzed hydrogen water (EHW). ChIP-X Enrichment Analysis 3 (ChEA3) was used for the enrichment analysis of transcription factors. In the ChEA3 analysis, the top 10 with the smallest p-value at p < 0.05 were selected and visualized. From Figure 4, the transcription factor Gut-Enriched Krueppel-Like Factor (KLF4), which is related to the enhancement of the skin barrier function (intestinal barrier function), was enriched as a transcription factor targeting the differentially expressed genes of electrolyzed hydrogen water. KLF4 has also been suggested to function as a colorectal cancer suppressor.

[0036] Figure 5 is a Volcano plot showing the expression variation of microRNA by electrolyzed hydrogen water (EHW) based on purified water (ACW) in human colorectal cancer cells (Caco-2 cells). The vertical axis in Figure 5, similar to Figure 1, indicates the statistical significance level, and the higher the numerical value, the more statistically significant it is. The horizontal axis in Figure 5 indicates the expression level of microRNA by electrolyzed hydrogen water. Values smaller than 0 indicate microRNAs with decreased expression levels, while values larger 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 at p < 0.05 in FDR). Among the 50, 27 are differentially expressed genes showing increased expression of microRNA (hsa-miR-148a-3p, hsa-miR-215-5p, hsa-miR-192-5p, hsa-miR-27b-3p, hsa-miR-3529-3p, hsa-miR-206, etc.). On the other hand, 23 are differentially expressed genes showing decreased expression of microRNA (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, etc.).

[0037] Figure 6 is a Heatmap showing the amount of variation (degree of variation) based on purified water (ACW) for microRNAs whose expression was varied by electrolyzed hydrogen water (EHW) in human colorectal cancer cells (Caco-2 cells), indicated by the shade of color, etc. The Heatmap was obtained by standardizing and comparing two sets of independent experimental data. Note that the English letters in Figure 5 represent gene names. In Figure 5, the upper part shows microRNAs with increased expression in electrolyzed hydrogen water (hsa-miR-192-5p~hsa-miR-200a-5p), and the lower part shows microRNAs with decreased expression in electrolyzed hydrogen water (hsa-miR-194-5p~hsa-miR-182-5p).

[0038] <Gene prediction and construction of interaction network> Based on TargetScan (https: / / www.targetscan.org / vert_72 / ), the targets of microRNA were predicted from the genes with expression variations, and an mRNA - microRNA related network was constructed. TargetScan refers to the microRNA target prediction algorithm and its database. Also, MCODE was used to identify the hub genes within the interaction network. MCODE refers to a method for discovering protein complexes from the protein - protein interaction (PPI) network. The results are shown in Figure 7.

[0039] Figure 7 shows the interaction analysis of mRNA - microRNA by TargetScan and MCODE in human colorectal cancer cells (Caco - 2 cells). Specifically, Figure 4 shows that using TargetScan, the mRNAs that are targets of the microRNAs that vary by electrolyzed hydrogen water (EHW) were screened from the genes with expression variations of electrolyzed hydrogen water, and furthermore, using MCODE, the results of analyzing the protein - protein interaction (PPI) network, in other words, the network is shown. From Figure 7, CUL5 (Cullin 5), GOLGA7 (Golgin A7), has - miR - 429 and has - miR - 200c - 3p were selected as hub genes. Note that CUL5 and GOLGA7 are genes with expression variations showing increased expression. On the other hand, has - miR - 429 and has - miR - 200c - 3p are genes with expression variations showing decreased expression.

[0040] <Prediction and selection of hub genes controlling transcription factors (TFs)> Transcription factors (TFs) were regarded as genes when finding mRNA - microRNA interactions. The pairs of TF - mRNA and TF - microRNA are from the following three transcription factor databases: ·hTFtarget (https: / / ngdc.cncb.ac.cn / databasecommons / database / id / 6946) ·motifmap (http: / / motifmap.ics.uci.edu / ) ·TransmiR v2.0 (http: / / www.cuilab.cn / transmir) was used to obtain the results, which are shown in Fig. 8.

[0041] Fig. 8 shows the results of analyzing the transcription factors common to the hub genes responding to electrolyzed hydrogen water (EHW) by utilizing the above three transcription factor databases in human colorectal cancer cells (Caco-2 cells). Specifically, Fig. 8 shows the results of analyzing the transcription factors common to the four hub genes (CUL5, GOLGA7, has-miR-429, has-miR-200c-3p) identified above. From Fig. 8, through the collation of the above three transcription factor databases, 16 transcription factors shown in Table 1 below were found as the common transcription factors controlling the four hub genes of CUL5, GOLGA7, has-miR-429, and has-miR-200c-3p. Among the transcription factors, especially "KLF4" (Krueppel-Like Factor 4) is considered highly likely to be involved in the regulation of CUL5, GOLGA7, has-miR-429 and / or has-miR-200c-3p in intestinal epithelium.

[0042]

Table 1

[0043] <Summary> Through the above analysis, four response genes, CUL5 (Cullin 5), GOLGA7 (Golgin A7), has-miR-429, and has-miR-200c-3p, were identified as the hub genes responding to electrolyzed hydrogen water with a dissolved hydrogen concentration of 1056 ppb or more and 1080 ppb or less. The electrolyzed hydrogen water of the present invention acts on enhancing the intestinal barrier maintenance function by immune regulation by regulating the up-regulation or down-regulation of the expression of these response genes, that is, it has the activity of enhancing the intestinal barrier maintenance function.

Industrial Applicability

[0044] As an application example of the present invention, electrolyzed hydrogen water having an activity of enhancing the intestinal barrier maintaining function can be mentioned.

Claims

1. By regulating the expression of the response gene, it has the activity of enhancing the intestinal barrier maintenance function, The electrolyzed hydrogen water, characterized in that 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. The electrolyzed hydrogen water according to claim 1, characterized in that the dissolved hydrogen concentration is 1056 ppb or more and 1080 ppb or less.

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