Molecular Hydrogen Inhalation: Effects on Health, Exercise Capacity and Inflammatory Response - in Vivo/in Vitro Studies

1\. RESEARCH PROJECT OBJECTIVES 1.1. Research project objectives The main aim of the project is to comprehensively evaluate the impact of molecular hydrogen inhalation on exercise capacity, inflammatory response, anti-tumour potential of human blood serum, and related molecular-genetic mechanisms involving oxidative stress, iron metabolism, gene expression regulation, and intracellular signalling pathways, assessed through in vivo and in vitro analyses.

The project aims to:

1. Demonstration relationship between oxidative stress parameters, uric acid concentration and nitric oxide degradation products in groups of people subjected to two weeks of daily inhalation with the use of a molecular hydrogen generator. 2. Indicate of whether and how one time and daily inhalation with the use of a molecular hydrogen generator will affect BDNF and hepcidin concentration, and whether these changes will be accompanied by greater resistance of skeletal muscles to damage induced by exercises; 3. Show whether two weeks daily inhalation with the use of a molecular hydrogen generator will induce a higher changes of BDNF, hepcidin erytropheron (ERFE) and erythropoietin (EPO) concentration (compared to a single session of inhalation with the use of a molecular hydrogen); 4. Show how daily inhalation with the use of a molecular hydrogen generator and physical activity procedures effects human serum anti-tumour potential, and is it associated with Vitamin D status and Iron metabolism. 5. Show is there a relationship between one time and two weeks of daily inhalation using a molecular hydrogen generator and the expression of genes related to the stress response (HSF-1, NF-kB, TNF-dependent pathway). 6. Show the relationship between the expression of genes related to muscle cell growth (e.g. myostatin gene) and the effect of molecular hydrogen inhalation? 7. Demonstrate the relationship between the expression of genes related to intracellular metabolism and the effect of molecular hydrogen inhalation. 8. Show the relationship between the expression of genes encoding enzymes of energy pathways (e.g. GAPDH, LDH) and the effect of molecular hydrogen inhalation in the study group 9. Show the molecular hydrogen inhalation effects on the expression of genes responsible for membrane transport and the hedgehog pathway in muscle cells (including Gli1), expression of transcription factors: Hif-1-alpha (hypoxia-inducible factor) and NF-kB and selected genes dependent on their activity.

1.2. Hypotheses

In the project on the basis of current knowledge, the following hypotheses are stated:

1. Daily inhalation with the use of a molecular hydrogen will increase the concentration of BDNF, which will correlate with higher skeletal muscle resistance to damage; 2. Two week daily inhalation with the molecular hydrogen will increase the concentration of BDNF, which will persist over a longer period compering to a single inhalation with the molecular hydrogen session; 3. Daily inhalation with the molecular hydrogen and physical activity effects on vitamin D binding protein, megalin, cubilin concentration changes. 4. Daily inhalation with the molecular hydrogen protects muscles and reduces oxidative stress induced by physical exercise and protects against oxidative stress induced by high Iron concentration (lower inflammation process and cfDNA concentration). 5. Daily inhalation with the molecular hydrogen effects human serum anti-tumor potential against human LNCaP prostate cancer cells.

2\. SIGNIFICANCE OF THE PROJECT Physical performance, encompassing endurance, muscle strength, and explosive power, serves as a fundamental element for success in sports among both non-athletic individuals and athletes.

It not only enhances competitive performance on the field but also encourages healthy adults to engage in sports. Oxidative stress arises when the products of oxygen metabolism accumulate and exceed the body's ability to resist oxidation. Research indicates that physical activity at varying intensities can influence the levels of different oxidative biomarkers.

Nevertheless, engaging in physical exercise, particularly at moderate to high intensities, may result in excessive oxidative stress, adversely affecting redox homoeostasis, increasing fatigue, and ultimately diminishing physical performance.

Consequently, there has been a focus on investigating potential antioxidant strategies to develop effective methods for improving physical performance. Molecular hydrogen (H2) has emerged as a promising antioxidant that selectively neutralises hydroxyl radicals and peroxynitrite in cells without diminishing other reactive species.

The suggested benefits of molecular hydrogen in the body, are due the fact that H2 is a potent antioxidant and signalling molecule. Unlike conventional antioxidants that non-selectively scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS), hydrogen selectively targets highly reactive and deleterious species such as hydroxyl radicals (•OH) and peroxynitrite (ONOO-), thereby preserving ROS involved in physiological signalling, which is crucial for cellular homoeostasis and adaptation. Recent studies have shown that modulating ROS and subsequently regulating the gas transmitters nitric oxide and carbon monoxide to influence NO-CO metabolism may have beneficial effects on various diseases. Studies have shown that molecular hydrogen, which can be delivered to the body in various forms (i.e. inhaled gas - H2 inhalation, drinking hydrogen-rich water (HRW) and intravenously in H2 saline solution), can penetrate cell membranes and rapidly diffuse into cellular organelles (e.g. mitochondria), thereby increasing the functional efficiency of mitochondria and enhancing ATP production or lactate oxidation. Recent human studies have begun to investigate the potential advantages of H2 for enhancing physical performance, showing significant promise for H2-based interventions. However, the findings and study designs regarding the impact of H2 on physical performance have varied.

Regulation of gene expression: ROS play a role in the regulation of gene expression, particularly in genes involved in stress responses and antioxidant production. By maintaining physiological levels of ROS, hydrogen can aid in the proper regulation of these genes, supporting cellular defence mechanisms and resilience against oxidative stress. Overall, these beneficial properties not only protect cells from oxidative damage but also support critical signalling pathways that promote health, adaptation, and recovery.

The potential effects of molecular hydrogen on exercise physiology are multifaceted, influencing a wide range of physiological processes encompassing energy metabolism, oxidative stress modulation, inflammation regulation, cell signalling modulation, and recovery facilitation mechanisms. By regulating these key pathways, molecular hydrogen offers an alternative approach to optimising athletic performance, mitigating exercise-induced muscle damage and accelerating post-exercise recovery.

Despite growing interest in this area, several fundamental questions remain unanswered\]. A primary concern is determining which specific exercise patterns-aerobic, anaerobic derive the most benefit from molecular hydrogen inhalation. It is well established that sports encompass a diverse range of activities, each requiring distinct skills such as cardio respiratory fitness, muscular strength, muscular endurance, flexibility, agility, coordination, power, reaction time, and speed. However, the extent to which HRW can enhance these specific skills remains unclear and warrants further investigation. Additionally, to effectively develop of molecular hydrogen inhalation procedure as improving sport result procedure and promote its use among a broader athletic audience, it is crucial to identify the specific physiological advantages that might enhance athletic performance.

Recent years brought the era of cell-free DNA (cfDNA) as a potential biomarker of the level of injury, which is gaining interest in many various biomedical disciplines, including the field of exercise physiology. There are more and more studies looking for the role of cfDNA as a potential hallmark of the overtraining syndrome in: resistance training, marathon run, continuous treadmill running, incremental exercise, rowing exercise, strength training.

Besides cfDNA might be related to, or trigger adaptations of immune function induced by strenuous exercise. Publication from 2017 revealed an increase cf-DNA level even during aerobic running below the lactate steady state depending on intensity and duration. It was observed that cfDNA concentrations peaked immediately after acute exercise and about 1h post-exercise returned to baseline levels. Furthermore, typical markers of skeletal muscle damage (C-reactive protein, uric acid, creatine kinase, myoglobin) display delayed kinetics compared with the cfDNA peak response. All of that underlines the potential of cfDNA as a biomarker for exercise load in both-the aerobic and the anaerobic state. However, there was not much effort put into finding the correlations between cf-DNA and iron state and exercises induced inflammation process.

This project will have an impact on the development of the current state of knowledge of the mechanisms of biochemical response to specific tissue affecting method in form of a molecular hydrogen inhalation and will allow to determine its role on the post-exercise response in form of changes in biochemical markers secretion, expression of selected trophic factors and changes in iron metabolism in this process. Moreover, this project will try to take into account the role of hepcidin, vitamin D and cfDNA. Additionally, the present project may contribute to the determination of the role of presented inhalation procedure on cells proliferation, as example of ant-tumour proprieties, regulation of the expression of genes related to the stress response (HSF-1, NF-kB, TNF-dependent pathway), muscle cell growth (e.g. myostatin gene), energy pathways (e.g. GAPDH, LDH) and the membrane transport and the hedgehog pathway ls (including Gli1), Hif-1-alpha and NF-kB.

Therefore, the results of this project could be a significant step in expanding our understanding of the role of molecular hydrogen inhalation in the healthy population (in modulating post-exercise muscle damage, cells proliferation, inflammation process) and the role of iron metabolism and vitamin D in those changes. In addition, the knowledge on the protective effect of molecular hydrogen inhalation procedures and their relation to BDNF changes and protein kinase activity will give us overview of its novel activity and use.

The research team involved in this project possesses extensive expertise and significant experience confirmed by numerous scientific publications, particularly in the field of human physiology, exercise-induced adaptive responses, and various intervention strategies aimed at optimising recovery and performance. Members of the team have previously published research on oxidative stress modulation, inflammatory responses, genetic expression changes, and biochemical adaptations following physical exercise, ensuring that the methodologies and analytical approaches proposed in this study are robust, reliable, and scientifically validated

3\. WORK PLAN In order to achieve the objectives of the study the minimal population sample size allowing to support the appropriate power of the study of interactions between the effects was calculated using GPower ver. 3.1.9.2 software. The minimal sample size was 64 people. Due to the possibility of participants not completing the project, a 20% population increase was assumed in order to obtain the desired study size population of 80 people will be recruited, then randomly divided into two groups: Experimental (N=40) and Control (N=40).

Furthermore, groups will be d

• Measurement of proteins involved in antioxidant defence using Western Blotting Techniques [Срок оценки: Before and after 1-time and 14-times inhalation using a molecular hydrogen generator]
• Messurment of cfDNA changes [Срок оценки: Before, and 5 min, 60 min after every WAnT and Bruce Test performance]
• Measurement of isolated serum cytotoxic activity [Срок оценки: Up to 12 months post-experimental period]