Authors:
Ono, H.; Nishijima, Y.; Adachi, N.; Sakamoto, M.; Kudo, Y.; Kaneko, K.; Nakao, A.; Imaoka, T.

Source: Medical Gas Research, Volume 2, Article 21 (2012)

Methodology

This pilot study evaluated the safety of molecular hydrogen (H₂) inhalation in patients with acute cerebral ischemia. In three patients, hydrogen concentration (HC) in arterial and venous blood was measured using gas chromatography before, during, and after inhalation of 3% or 4% H₂ gas. Simultaneously, basic physiological parameters were monitored. In a separate consistency check, hydrogen levels were measured in venous blood from ten additional patients at the end of a 30-minute H₂ inhalation session.

Results

• Stable blood H₂ levels were reached approximately 20 minutes after the start of inhalation.

• After cessation, HC levels dropped rapidly—within about 6 minutes in arterial blood and 18 minutes in venous blood—to 10% of peak levels.

• Physiological parameters (e.g., blood pressure, heart rate, oxygen saturation) remained unchanged in all patients during H₂ inhalation, supporting its safety.

• In the consistency study, HC levels varied between patients but improved with better instruction and encouragement during inhalation.

Conclusion

Inhalation of molecular hydrogen at concentrations of at least 3% for 30 minutes achieves sufficient blood H₂ levels comparable to those seen in animal studies, without adversely affecting vital physiological parameters. However, optimizing delivery methods is important for achieving consistent results in clinical settings.

Citation

Ono H, Nishijima Y, Adachi N, et al. (2012). A basic study on molecular hydrogen (H₂) inhalation in acute cerebral ischemia patients for safety check with physiological parameters and measurement of blood H₂ level. Medical Gas Research, 2:21. https://doi.org/10.1186/2045-9912-2-21

Authors:
Botek, M.; Sládecková, B.; Krejčí, J.; Pluháček, F.; Najmanová, E.

Source: Acta Gymnica, Volume 51 (2021), Article e2021.009
https://doi.org/10.5507/ag.2021.009

Background

Hydrogen-rich water (HRW) has been shown to exhibit stimulating effects on the human body. However, its impact on the autonomic nervous regulation of the heart in resting conditions remains underexplored.

Objective

To assess the effect of acute HRW ingestion on cardiac autonomic regulation during a 50-minute seated rest period in healthy young women.

Methods

• Study Design: Double-blind, randomized, placebo-controlled crossover trial.

• Participants: 14 healthy females (mean age: 21.7 ± 1.2 years; body weight: 67.8 ± 8.7 kg; height: 167 ± 5.5 cm).

• Intervention: Participants ingested 1260 mL of either HRW or placebo.

• Measurement: Heart rate variability (HRV) was monitored during seated rest using time-domain indices:

  • RMSSD (Root Mean Square of Successive Differences)

  • SDNN (Standard Deviation of NN intervals)

  • SDNN/RMSSD ratio (sympatho-vagal balance index)
    These values were log-transformed (Ln) for statistical analysis.

Results

• A significant increase in the Ln SDNN/RMSSD ratio was observed 25 minutes (HRW: 0.40 ± 0.30 vs. placebo: 0.26 ± 0.25, p = 0.049) and 35 minutes (HRW: 0.44 ± 0.30 vs. placebo: 0.28 ± 0.28, p = 0.029) after ingestion, indicating increased sympathetic activity.

• Ln SDNN was significantly higher in the HRW group at 45 minutes (HRW: 4.41 ± 0.42 ms vs. placebo: 4.28 ± 0.31 ms, p = 0.049).

• No significant effect on vagal (parasympathetic) activity (RMSSD) was observed.

Conclusion

Acute ingestion of hydrogen-rich water stimulates relative sympathetic activation between 25 and 35 minutes after consumption in healthy females during resting seated conditions, without altering vagal tone.

Authors:
Li, XZ.; Li, LT.; Liu, XC.; Wu, JW.; Sun, XY.; Li, ZL.; Geng, YJ.; Liu, FL.; Zhou, YJ.

Source: Current Molecular Medicine, Volume 19, Issue 4 (2019), pp. 294–302.

Background

Hydrogen (H₂) has been demonstrated to exert bioactive effects on cardiac tissue. Ischemia-reperfusion (I/R) injury remains a significant challenge in cardiovascular medicine due to oxidative stress and cellular apoptosis.

Objective

To evaluate the protective effect and underlying mechanisms of hydrogen-rich water (HRW) in a rat model of myocardial ischemia-reperfusion injury, particularly focusing on key signal transduction pathways.

Methods

• Subjects: 20 male Wistar rats.

• Procedure: Rats were subjected to myocardial ischemia-reperfusion injury and treated with hydrogen-rich water.

• Sample Collection: Left ventricular tissue was harvested for analysis.

• Analytical Techniques:

  • Protein expression profiling using protein microarrays.

  • KEGG pathway enrichment via gene ontology (GO) analysis.

  • Western blotting and immunohistochemistry for expression of JAK2, STAT1, STAT3, and their phosphorylated forms (p-JAK2, p-STAT1, p-STAT3).

  • TUNEL assay to detect apoptotic cells in myocardial tissue.

Results

• Expression of 25 proteins across five signaling pathways was reduced in the HRW-treated group compared to control.

• Phosphorylation levels of JAK2 and STAT3 (p-JAK2/JAK2, p-STAT3/STAT3) were increased, while p-STAT1/STAT1 was decreased in the HRW group.

• Apoptosis rates in cardiomyocytes were significantly reduced following HRW treatment.

Conclusion

Hydrogen-rich water effectively reduces myocardial ischemia-reperfusion injury by modulating the JAK2-STAT3 signaling pathway, thereby inhibiting apoptosis in cardiomyocytes. These findings highlight the potential of HRW as a cardioprotective therapeutic strategy.

Authors:
Li, LT.; Liu, TT.; Liu, L.; Zhang, Z.; Li, SC.; Zhang, ZL.; Zhou, YJ.; Liu, FL.

Source: Journal of Bioenergetics and Biomembrane, Volume 52, Issue 4 (2020), pp. 257–268.
DOI: 10.1007/s10863-020-09835-7

Objective

To evaluate the impact of hydrogen-rich water (HRW) on the myocardial metabolism in a rat model of myocardial ischemia-reperfusion injury (MIRI) using metabolomic analysis.

Methods

• Sample: 12 rats randomly divided into a control group and a hydrogen-rich water group (6 rats each).

• Perfusion Setup: After heart excision, hearts were mounted on a Langendorff apparatus and perfused with oxygenated solutions at 37°C.

  • Control group: perfused with Krebs–Ringer solution.

  • HRW group: perfused with Krebs–Ringer + hydrogen-rich water.

• Analytical Technique:

  • Liquid Chromatography–Mass Spectrometry (LC-MS) for untargeted metabolomics.

  • Statistical Analyses:

    • Principal Component Analysis (PCA)

    • Partial Least Squares Discriminant Analysis (PLS-DA)

    • Orthogonal PLS-DA (OPLS-DA)

    • Variable Importance in Projection (VIP, threshold ≥ 1)

    • Independent-sample T-tests (p < 0.05)

• Targeted Pathways: Focus on glycerophospholipid metabolism and other relevant pathways.

Results

• Seven metabolic pathways significantly altered by HRW treatment, including:

  • Glycerophospholipid metabolism

  • Glycosylphosphatidylinositol (GPI)-anchor biosynthesis

  • Purine metabolism

• Ten biomarkers were significantly different between groups, including:

  • Phosphatidylcholine

  • Phosphatidylethanolamine

  • Phosphatidylserine

Conclusion

Hydrogen-rich water modulates metabolic imbalances in ischemia-reperfusion injured myocardial tissue and may alleviate MIRI in isolated rat hearts through multiple signaling pathways. These findings underscore HRW’s potential as a cardioprotective intervention by restoring key aspects of myocardial metabolism.

Authors:
Li, LT.; Li, XZ.; Zhang, Z.; Liu, L.; Liu, TT.; Li, SC.; Liu, S.; Zhou, YJ.; Liu, FL.

Source: Current Molecular Medicine, Volume 20, Issue 5 (2020), pp. 396–406.
DOI: Not specified in original, but source journal available here.

Objective

To investigate the effect of hydrogen-rich water (HRW) on PI3K/AKT-mediated apoptosis in a rat model of myocardial ischemia-reperfusion injury (MIRI).

Methods

• Subjects: 60 rats randomly assigned to either the HRW-treated group or a control group.

• Perfusion Setup:

  • All hearts were excised and fixed using a Langendorff apparatus.

  • Control: perfused with Krebs–Ringer solution (K-R).

  • Treatment: perfused with K-R + hydrogen-rich water.

• Time Points: Each group was subdivided into three subgroups:

  • Pre-ischemia, ischemia, and reperfusion (10 rats each).

  • Procedures: reverse perfusion (10 min), normal treatment (20 min), reperfusion (20 min).

• Measurements:

  • mRNA and protein expression levels of PI3K, AKT, p-AKT, FoxO1, Bim, and caspase-3 were measured by:

    • RT-qPCR

    • Western blotting

    • Immunohistochemistry (IHC)

  • Caspase-3 activity was analyzed by spectrophotometry.

Results

• Activation of PI3K/AKT Pathway:

  • Significantly higher expression of PI3K, AKT, and p-AKT in the HRW group during the reperfusion phase.

• Suppression of Apoptotic Markers:

  • HRW group showed significantly lower levels of FoxO1, Bim, and caspase-3 (both mRNA and protein) compared to the control, particularly during the ischemia-reperfusion phase.

• Conclusion:
  Hydrogen-rich water activates the PI3K/AKT pathway, thereby attenuating myocardial ischemia-reperfusion injury and inhibiting cardiomyocyte apoptosis in isolated rat hearts.

Authors:
Li, LT.; Liu, TT.; Li, XZ.; Liu, XC.; Liu, L.; Li, SC.; Li, ZL.; Zhou, YJ.; Liu, FL.

Source:
International Journal of Medical Sciences, Volume 16, Issue 9 (2019), pp. 1254–1259.
DOI: 10.7150/ijms.35984

Objective

To analyze differentially expressed proteins (DEPs) involved in the protective effect of hydrogen-rich water (HRW) on myocardial ischemia-reperfusion injury (MIRI) and to examine their associated biological processes and signaling pathways using bioinformatic methods.

Methods

• Subjects: 20 Wistar rats were randomly and equally divided into:

  • Control group: Perfused with Krebs–Ringer solution (K-R)

  • HRW group: Perfused with K-R + hydrogen-rich water

• Procedure:
  Hearts were excised and mounted on a Langendorff apparatus for perfusion.

• Protein Profiling:

  • Proteins were extracted from ventricular tissues

  • A high-throughput protein chip (GSR-CAA-67) was used to identify DEPs

• Bioinformatics:

  • Gene Ontology (GO) enrichment analysis identified 359 biological processes

  • KEGG pathway enrichment analysis identified five key signaling pathways

Results

• Protein Expression:

  • 25 proteins were found to be significantly downregulated (P < 0.05) in the HRW-treated group compared to control.

• Biological Processes Identified:

  • Regulation of signaling pathways

  • Immune responses

  • Formation of cardiovascular endothelial cells

• Key Pathways Identified (via KEGG):

  • Specific details not listed here, but five distinct signaling pathways were enriched.

Conclusion

Hydrogen-rich water modulates the expression of 25 key proteins, influencing multiple biological processes and metabolic pathways that are associated with reduction of myocardial ischemia-reperfusion injury. These findings provide theoretical and mechanistic evidence supporting the clinical application of hydrogen-rich water in cardiac protection.

Authors:
Feng, R.; Cai, M.X.; Wang, X.D.; Zhang, J.J.; Tian, Z.J.

Source:
Applied Biochemistry and Biotechnology, Volume 187, Issue 3 (2019), pp. 663–676
DOI: 10.1007/s12010-018-2841-0

Background

Hydrogen-rich saline (HRS) is known to reduce oxidative stress, and early aerobic exercise (eAE) is recognized as an effective exercise preconditioning (EP) strategy against ischemia-reperfusion (I/R) injury. However, whether combining eAE with HRS (eAE-HRS) provides enhanced protection against myocardial injury induced by acute myocardial infarction (AMI) was previously unclear.

Objective

To evaluate the preventive effect of eAE-HRS on AMI-induced myocardial injury in rats and explore the underlying protective mechanisms, particularly those related to oxidative stress and mitochondrial function.

Methodology

• Subjects: Sprague-Dawley rats

• Interventions:

  • HRS (1.6 ppm) administered orally at 10 ml/kg/day for 3 weeks

  • 3-week treadmill training as aerobic exercise (eAE)

  • MI induced by ligation of the left anterior descending coronary artery

• Groups:

  • HRS only

  • eAE only

  • eAE-HRS combined

  • Control (no intervention)

• Assessments:

  • Cardiac function metrics: LV systolic pressure (LVSP), end-diastolic pressure (LVEDP), ±dp/dt(max), cardiac coefficient, pH

  • Injury biomarkers: CK-MB, c-TnI, h-FABP, infarct size

  • Oxidative stress markers: SOD, total antioxidant capacity (T-AOC), MDA, catalase

  • Protein expression: OGG1 (DNA repair), Tom20, Tom40, Tim23 (mitochondrial transport proteins)

Results

• Functional Improvement:

  • eAE-HRS improved LV systolic function, reduced diastolic pressure, and enhanced contraction/relaxation rates.

• Reduction of Injury:

  • Lowered serum markers (CK-MB, c-TnI, h-FABP)

  • Decreased infarct size

• Antioxidant Enhancement:

  • Increased SOD and T-AOC

  • Decreased MDA and catalase levels

• Mitochondrial and Antioxidant Protein Upregulation:

  • Elevated OGG1, Tom20, Tim23 expression in myocardium

  • Tom40 expression remained unchanged

Conclusion

The combination of early aerobic exercise and hydrogen-rich saline (eAE-HRS) offers significant cardioprotective effects against AMI-induced myocardial injury. The effect is likely mediated by modulating mitochondrial-related proteins and enhancing antioxidant defense mechanisms. eAE-HRS thus holds potential as a novel preconditioning strategy to prevent cardiac damage after AMI.

Authors:
Li, L.T.; Liu, T.T.; Liu, L.; Li, S.C.; Zhang, Z.; Zhang, R.S.; Zhou, Y.J.; Liu, F.L.

Source: Journal of Bioenergetics and Biomembranes, Volume 51, Issue 6, Pages 393–402 (2019)

Objective

This study examined the effects of hydrogen-rich water (HRW) on oxidative stress through the activation of the Nrf2/ARE signaling pathway in a rat model of myocardial ischemia-reperfusion injury (MIRI).

Methodology

Sixty rats were randomly assigned to either a hydrogen-rich water group or a control group (30 rats each). Each group was further divided into three subgroups: pre-ischemic phase, ischemic phase, and reperfusion phase. After the hearts were excised, they were mounted on a Langendorff apparatus and perfused with oxygenated perfusate at 37°C. The control group received Krebs–Ringer (K–R) solution, while the HRW group was perfused with K–R solution containing hydrogen-rich water.
Expression levels of mRNA and protein for Nrf2, NQO1, HO-1, and SOD-1 in cardiomyocytes were measured using RT-qPCR, immunohistochemistry (IHC), and Western blot analysis. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were also assessed.

Results

HRW significantly enhanced the activation of the Nrf2/ARE pathway, with increased mRNA and protein levels of Nrf2, NQO1, HO-1, and SOD-1 during the ischemia-reperfusion phase compared to the ischemic phase (P < 0.05). In contrast, these levels were significantly reduced in the control group during reperfusion compared to ischemia.
During the ischemia-reperfusion phase, the HRW group exhibited significantly elevated SOD activity and reduced MDA content, while the control group showed decreased SOD activity and increased MDA levels (P < 0.05).

Conclusion

Hydrogen-rich water may alleviate myocardial ischemia-reperfusion injury by activating the Nrf2/ARE signaling pathway and reducing oxidative stress in myocardial tissues.

Citation

Li, L., Liu, T., Liu, L., et al. (2019). Effect of hydrogen-rich water on the Nrf2/ARE signaling pathway in rats with myocardial ischemia-reperfusion injury. Journal of Bioenergetics and Biomembranes, 51(6), 393–402. https://doi.org/10.1007/s10863-019-09814-7

Authors:
Liu, Y.; Wang, D.F.; Chen, X.J.; Yuan, J.; Zhang, H.L.; Fu, J.Q.; Wang, Y.; Lan, Q.

Source: International Journal of Clinical and Experimental Pathology, Volume 10, Issue 3, Pages 3807–3815 (2017)

Background

Traumatic brain injury (TBI) is a leading cause of mortality and morbidity worldwide and results in severe long-term disabilities.

Objective

This study aimed to evaluate the effect of hydrogen-rich water (HRW) on angiogenesis in the brain tissue surrounding lesions in rats subjected to TBI.

Methodology

Fifty-four adult male Sprague-Dawley rats were randomly assigned to three groups: sham-operated, TBI, and TBI + HRW. After inducing TBI, neurological severity scores (NSS) were assessed. Hematoxylin-eosin staining, immunohistochemistry, Western blotting, and reverse transcription polymerase chain reaction (RT-PCR) were used to analyze brain tissue at various time points post-injury.

Results

On days 3 and 7 post-injury, NSS scores in the TBI + HRW group were significantly lower compared to the TBI group (P < 0.05). At 24 hours and 3 days post-injury, histopathological analysis of the lesion boundary tissue in TBI rats revealed hemorrhagic necrosis, severe brain edema, and disrupted neuronal architecture, with the most pronounced changes observed on day 3. Compared to the TBI group, the TBI + HRW group had reduced brain edema. On day 7, neonatal capillary hyperplasia was significantly greater in the TBI + HRW group than in the TBI group (P < 0.01).
At all time points, expression levels of HIF-1α and VEGF protein and mRNA were significantly higher in the TBI + HRW group than in the TBI group (P < 0.01 / P < 0.05), and both were significantly higher than in the sham group (P < 0.01).

Conclusion

Hydrogen-rich water promotes angiogenesis and improves neurological function following TBI by upregulating HIF-1α and VEGF expression. These findings suggest HRW as a promising adjunct for enhancing brain repair after injury.

Citation

Liu, Y., Wang, D., Chen, X., Yuan, J., Zhang, H., Fu, J., … & Lan, Q. (2017). Effect of hydrogen-rich water on the angiogenesis in lesion boundary brain tissue of traumatic brain injury-challenged rats. International Journal of Clinical and Experimental Pathology, 10, 3807–3815.