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

Summary To comprehensively address the research aims and explore the 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 anty-tumor 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. 1.1. Primary Objectives 1. Demonstration relationship between post-exercise oxidative stress parameters, and two weeks of daily inhalation with the use of a molecular hydrogen generator. 2. Indicate of whether and how daily inhalation with the use of a molecular hydrogen generator will affect BDNF and hepcidin, erytropheron (ERFE) and erythropoietin (EPO). 3. Show how daily inhalation with the use of a molecular hydrogen generator and physical activity procedures effects human serum anti-tumor potential, and is it associated with Vitamin D status and Iron metabolism. 1.2. Secondary Objectives 1. Examine the relationship between 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). 2. Show the relationship between the expression of genes related to muscle cell growth (e.g. myostatin gene), intracellular metabolism, energy pathways (e.g. GAPDH, LDH) and the effect of molecular hydrogen inhalation? 3. 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. 1.3. 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. III. Research project methodology In order to achieve the objectives of the study 80 people will be recruited, then randomly divided into two groups: Experimental ((N=40) and Control (N=40). Furthermore, groups will be divided into subgroups to implement the assumptions of one-time and 10-time inhalation with the use of a molecular hydrogen generator on the level of induced muscle damage and the level of maximum anaerobic and aerobic capacity. The whole study will be carried out using the assumptions of the experiment - a blind test (inhalation with normal air but under the use of H2 generator (switched of), and in accordance with the principles of the experiment. In the study an experiment based on an ex post facto research plan will be used, due to the lack of manipulation of the grouping variable. Study will be based on comparative analysis and regression analysis. In the independent variable test, the group (non-trainees), variables dependent on molecular hydrogen inhalation and the anaerobic/aerobic performance characteristics, biochemical blood indicators. Minimal (40 people each) sample size was calculated according to Kirby et all (2002) and Kadam and Bhalerao (2010). IV. The study will consist of twelve parts. For anaerobic power of the lower limbs measurement of double Wingate anaerobic test (WAnT) will be conducted on a cycle ergometer, * For the measurement of Aerobic Components of Fitness and post-aerobic exercises response Bruce Treadmill Test will be performed. * The blood collection for diagnostic tests will be strictly dependent on the requirements of a particular designation,

Sex

All

Minimum age

18 Years

Maximum age

49 Years

Inclusion Criteria:

The study will be used purposeful selection of the following criteria:

* age 18-25,
* not taking medicines during the study,
* negative history of cardiovascular disorders,
* negative history of autonomic nervous system disorders,
* negative history of mental disorders,
* negative history of cerebrospinal traumas,
* negative history of other diseases that may directly affect obtained results,
* good health status (no concurrent injuries),
* no drugs intake,
* no supplements consumption,

In the study as a not training grope age and morphologically appropriate participation will take part. Participants will be recruited basing on a voluntary letter of intent. All representatives of the analysed group participating in the pre-qualification research will fill in the physical activity sheet - Global Health Activity Questionnaire - World Health Organization in Polish adaptation. This will allow to eliminate people who report high levels of physical activity (similar to the level of sport training individuals).

Exclusion Criteria:

* taking medicines during the study,
* history of cardiovascular disorders,
* history of autonomic nervous system disorders,
* history of mental disorders,
* history of cerebrospinal traumas,
* history of other diseases that may directly affect obtained results,
* concurrent injuries,
* drugs intake,
* supplements consumption.

• BACKGROUNDChomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156-9. doi: 10.1006/abio.1987.9999. PMID 2440339
• BACKGROUNDSim M, Kim CS, Shon WJ, Lee YK, Choi EY, Shin DM. Hydrogen-rich water reduces inflammatory responses and prevents apoptosis of peripheral blood cells in healthy adults: a randomized, double-blind, controlled trial. Sci Rep. 2020 Jul 22;10(1):12130. doi: 10.1038/s41598-020-68930-2. PMID 32699287
• BACKGROUNDFarley JB, Stein J, Keogh JWL, Woods CT, Milne N. The Relationship Between Physical Fitness Qualities and Sport-Specific Technical Skills in Female, Team-Based Ball Players: A Systematic Review. Sports Med Open. 2020 Apr 15;6(1):18. doi: 10.1186/s40798-020-00245-y. PMID 32297147
• BACKGROUNDTimon R, Olcina G, Gonzalez-Custodio A, Camacho-Cardenosa M, Camacho-Cardenosa A, Martinez Guardado I. Effects of 7-day intake of hydrogen-rich water on physical performance of trained and untrained subjects. Biol Sport. 2021 Jun;38(2):269-275. doi: 10.5114/biolsport.2020.98625. Epub 2020 Oct 22. PMID 34079172
• BACKGROUNDBotek M, Khanna D, Krejci J, Valenta M, McKune A, Sladeckova B, Klimesova I. Molecular Hydrogen Mitigates Performance Decrement during Repeated Sprints in Professional Soccer Players. Nutrients. 2022 Jan 25;14(3):508. doi: 10.3390/nu14030508. PMID 35276867
• BACKGROUNDItoh T, Hamada N, Terazawa R, Ito M, Ohno K, Ichihara M, Nozawa Y, Ito M. Molecular hydrogen inhibits lipopolysaccharide/interferon gamma-induced nitric oxide production through modulation of signal transduction in macrophages. Biochem Biophys Res Commun. 2011 Jul 22;411(1):143-9. doi: 10.1016/j.bbrc.2011.06.116. Epub 2011 Jun 23. PMID 21723254
• BACKGROUNDHancock JT, Russell G. Downstream Signalling from Molecular Hydrogen. Plants (Basel). 2021 Feb 14;10(2):367. doi: 10.3390/plants10020367.