A Comprehensive Analysis of the Electrolytic Hydrogen Water Mechanism via a Feedforward Loop and its Functional Role in Intestinal Cells In Vitro

Electrolytic hydrogen water (EHW) plays a critical role in modulating cellular metabolism; yet, the underlying molecular mechanisms remain unclear. This study utilized next-generation sequencing (NGS) to assess mRNA and miRNA expression in EHW-treated Caco-2 cells. Bioinformatics analysis identified differentially expressed genes (DEGs) and pathways influenced by EHW and highlighted its involvement in the oxidative stress response and tight junction formation. Protein-protein interaction (PPI) network analysis of the DEGs identified first-neighbor genes, supporting the role of EHW in suppressing oxidative stress-related genes while also enhancing the expression of the TCEB2-CUL5-COMMD8 (ECS complex) genes, both of which converged on the HIF-1 signaling pathway. We also constructed an mRNA-miRNA competing endogenous RNA (ceRNA) network, which revealed four hub genes, two non-coding RNAs (miR-429 and miR-200c-3p) and two protein-coding RNAs (CUL5 and GOLGA7). These genes co-target the transcription factor KLF4 in Caco-2 cells, forming a TF-miRNA-gene network (TMGN). EHW treatment significantly decreased the levels of miR-429 and miR-200c-3p and stabilized CUL5 and GOLGA7 transcripts post-transcriptionally as compared to ACW. Concurrently, reduced miRNA expression weakened their pre-transcriptional competition with mRNAs for KLF4 binding, further enhancing CUL5 and GOLGA7 expression. Phenotypic assays confirmed that continuous EHW treatment promotes Caco-2 cell differentiation. This study underscores the regulatory role of EHW in intestinal cells via feed-forward loops (FFLs), offering novel insights into the molecular mechanisms and functions of EHW. HighlightsO_LIIdentification of Novel Key Regulatory Genes Modulated by Electrolytic Hydrogen Water (EHW) Treatment: PPI network analysis demonstrated that EHW downregulates mitochondrial oxidative metabolism-related genes while upregulating TCEB2-CUL5-COMMD8 (ECS complex) expression within the HIF-1 axis. C_LIO_LIConstruction of a ceRNA Network: By integrating transcriptome and miRNA sequencing data from EHW-treated samples, we assembled an associated network and identified four hub genes in intestinal cells within the mRNA-miRNA ceRNA network: miR-429, miR-200c-3p, CUL5, and GOLGA7. C_LIO_LINovel Mechanistic Insights of Post- and Pre-Transcriptional Regulation by EHW: We identified KLF4 as a key transcription factor regulating EHW hub genes and constructed a TF-miRNA-gene (TMGN) feed-forward loop (FFL) network, offering new insights into EHW biomarkers. Our analysis revealed that EHW reduces miR-429 and miR-200c-3p levels, thereby enhancing CUL5 and GOLGA7 expression through both pre-transcriptional and post-transcriptional regulation. C_LIO_LIPhenotypic Confirmation: Continuous EHW treatment shortened the time required for Caco-2 cell differentiation. C_LI