The Effect of Inhalation Using a Molecular Hydrogen Generator on the Level of Exercise Capacity and Inflammatory Response, Anti-cancer Potential of Human Blood Serum in the Light of 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,