Hydrogen-Rich Water Fights Exercise Fatigue Through a Novel Metabolic Pathway

Exercise-induced fatigue is a universal performance limiter and, when chronic, contributes to aging-related decline, neuropathy, and cardiovascular disease. While antioxidants have long been studied as countermeasures, clinical evidence remains inconsistent and overdose risks exist. Molecular hydrogen has emerged as a safer alternative, but its precise cellular mechanism has remained elusive—until now.

This study established a mouse model of exercise-induced fatigue using 4 weeks of weighted forced swimming in male C57BL/6 mice. Animals were divided into control, fatigue, and hydrogen-rich water (HRW, >1.5 ppm) groups. HRW was delivered ad libitum via nanobubble-generated water stored in aluminum bags and refreshed every 8 hours to maintain hydrogen concentration. Behavioral assessments included exhaustive treadmill testing and rotarod performance. Serum biomarkers (BUN, lactate, creatine kinase, MDA, SOD, glutathione peroxidase) were measured, and gastrocnemius and liver tissues underwent histological and immunohistochemical analysis.

HRW-treated mice showed significantly improved treadmill endurance and rotarod performance compared to the fatigue group. Serum fatigue markers—blood urea nitrogen, lactate, and creatine kinase—were meaningfully reduced, and gastrocnemius muscle histology showed less injury. Critically, UHPLC-quadrupole time-of-flight mass spectrometry metabolomics revealed that fatigue suppressed itaconate levels, and HRW restored them by upregulating IRG1 (immunoresponsive gene 1), the enzyme responsible for itaconate biosynthesis from the TCA cycle intermediate cis-aconitate. Elevated itaconate subsequently activated the Nrf2/HO-1 antioxidant axis, reducing oxidative stress markers including COX-2.

In vitro validation used differentiated C2C12 murine myotubes treated with either an IRG1 inhibitor (IRG1-IN, 5 μM) or the cell-permeable itaconate analog 4-octyl itaconate (4-OI, 250 μM) in hydrogen-rich media. Western blot and qPCR confirmed that HRW upregulated Nrf2 and HO-1 expression, and that this effect was modulated by IRG1 activity and itaconate availability. Mitochondrial ROS (MitoSOX), total ROS, and mitochondrial membrane potential (JC-1) assays further supported HRW's mitochondrial protective role.

These findings establish a novel IRG1-itaconate/Nrf2/HO-1 signaling axis as the mechanistic basis for hydrogen's anti-fatigue effects, linking immunometabolism to exercise physiology. The study opens potential therapeutic avenues for athletes, aging populations, and patients with fatigue-associated conditions, though human trials are needed to confirm translational relevance.