Hydrogen exerts antioxidant and cytostatic effects on C2C12 myoblasts

Oxidative stress plays a pivotal role in the progression of lifestyle-related diseases and thus its mitigation is essential for managing these conditions. Hydrogen gas (H 2 ) exhibits potent antioxidant properties and is thus a promising candidate for mitigating these diseases. Various pathologies, including lifestyle-related diseases, are known to impair skeletal muscle physiology and regeneration; however, the specific effects of H 2 on skeletal muscle remain largely unexplored. We developed a specialized cell culture system capable of maintaining a sealed H 2 environment. Using this device, we evaluated the antioxidant capacity of 2% H 2 and its effects on C2C12 cell proliferation. Furthermore, we investigated whether H 2 modulates cell alterations induced by high-glucose (HG) conditions. In C2C12 cells, H 2 significantly inhibited hydrogen peroxide-induced lipid peroxidation and attenuated HG-induced abnormalities in mitochondrial membrane potential. Under standard conditions, H 2 suppressed cell proliferation without altering mitochondrial membrane potential or inducing necrosis or apoptosis. Under HG conditions, cell proliferative capacity decreased, and cell death (necrosis/apoptosis) increased. Although H 2 prevented HG-induced mitochondrial membrane hyperpolarization, it failed to rescue cells from death. Notably, H 2 further suppressed cell proliferation under HG conditions compared with HG alone, without exacerbating cytotoxicity. These results suggest that H 2 possesses antioxidant and cytostatic properties in C2C12 cells without discernible cytotoxicity. This study offers fundamental insights into the therapeutic potential of H 2 for managing oxidative stress-related muscular pathologies.