Protective effect of energized structured water on bioenergetic function and oxidative stress in H9c2 cells

• ESW pretreatment significantly increased maximal and spare respiratory capacity in H9c2 cells. • ESW treatment increased the glycolytic ATP production without affecting mitochondrial and total ATP production. • ESW-treated cells showed improved viability and reduced ROS levels under H₂O₂-induced oxidative stress. • ESW upregulated 717 genes and downregulated 422 genes in H9c2 cells. • Gene Ontology analysis revealed ESW treatment significantly impacts biological processes related to cellular stress adaptation and energy metabolism regulation. The energized structured water (ESW) concept supposes water can be imbued with energetic frequencies or vibrations, improving its quality and providing various health benefits. While some studies claim benefits, the mechanisms and effects of ESW are not well understood. This study investigates the impact of ESW on cellular bioenergetic function and oxidative stress in H9c2 cells. H9c2 cells were pretreated with ESW or control water (CW) at equivalent dilutions. Mitochondrial function was assessed using the Agilent Seahorse XF Pro Analyzer. Cell viability and reactive oxygen species (ROS) production were evaluated under H 2 O 2 -induced oxidative stress conditions. Differential gene expression analysis and Gene Ontology (GO) enrichment were conducted to identify significantly affected genes and biological processes in ESW-treated H9c2 cells. Results demonstrated that ESW pretreatment significantly increased maximal and spare respiratory capacity in H9c2 cells. In addition, ESW treatment increased the glycolytic ATP production without affecting mitochondrial and total ATP production. ESW treatment enhanced cell viability and reduced ROS production in cells exposed to H 2 O 2 -induced oxidative stress. Based on differential gene expression analysis, 717 genes including Myh1, Akr1b8, Hmox1, Kcng1, Ugt1a6, Clu, Gaa, Ftl1, Hspb7 , and Gba1 were up-regulated and 422 genes including Hist1h2an, Slfn4, Rpl22l1, Polr2k, Il1rl1, Ndufb1, Atp5mkl1 were down-regulated by ESW compared to the controls. GO analysis indicated that ESW treatment significantly affects biological processes related to cellular stress response pathways. These findings suggest that ESW treatment may enhance cellular bioenergetics and stress resistance in H9c2 cells, potentially through modulation of gene expression related to stress responses and energy metabolism.