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,
age 18-25,
not taking medicines during the study,
negative history of cardiovascular disorders,
negative history of autonomic nervous system disorders,
taking medicines during the study,
history of cardiovascular disorders,
history of autonomic nervous system disorders,
history of mental disorders,
Crossover
Inhalation using a molecular hydrogen generator
Procedure/SurgeryHydrogen inhalation will be performed by using a hydrogen gas generator similar in terms of capabilities to the generator Hycellvator ET100 (Helix Japan, Co., Ltd., Tokyo, Japan). The apparatus will be generating 30.0 mL/s gas mixture, consisting of 68.0% hydrogen (hydrogen purity, 99.99%) and 32.0% of oxygen. All gases will be supplied through a nasal cannula connected to the gas generators. Although we could not measure directly the hydrogen and oxygen An average inspiratory flow rate will be adjusted to 500 mL/s at rest, the hydrogen concentration in the inspired gas must have been around 4.08% at most. Control population will have inhalation with the use of gas generator that has the same outer shape as the used generator to produce Placebo (30.0 mL/s, ambient air 400 m above sea level) consisting of 0.00005% of hydrogen and 20.9% of oxygen.
Measurement of the inflammation blood markers using Magnetic Luminex Performance Assays
The intake will be performed in appropriate standardized BD Vacutainer tubes by qualified medical personnel: Serum and plasma will be separated from the samples, Each sample to obtain the serum will be centrifuged at 2500-3000 rpm for 10 minutes in 4° C and stored in 1.5 ml tubes at - 80° C until the assay (no longer than 6 months). The following secretory factors and markers of inflammation will be determined in serum or blood plasma (depending on the requirements of the method used) and then analysed in detail using immu: • high sensitive C-protein (hsCRP), • creatine kinas (CK), • lactic acid (LA), • interleukin 6 (IL6), • interleukin 10 (IL10), • interleukin 15 (IL15),
Measurement of the Iron metabolism blood markers using Magnetic Luminex Performance Assays and ELISA Assays
The intake will be performed in appropriate standardized BD Vacutainer tubes by qualified medical personnel: Serum and plasma will be separated from the samples, Each sample to obtain the serum will be centrifuged at 2500-3000 rpm for 10 minutes in 4° C and stored in 1.5 ml tubes at - 80° C until the assay (no longer than 6 months). The following secretory factors and markers will be messured: • free Iron • hepcidin, • transferrin, • ferritin, • EPO, • EFRE, • TIBC,
Measurement of neurotrophic, angiogenic blood markers using Automated Hematology Analyzers and Magnetic Luminex Performance Assays
The intake will be performed in appropriate standardized BD Vacutainer tubes by qualified medical personnel: Serum and plasma will be separated from the samples, Each sample to obtain the serum will be centrifuged at 2500-3000 rpm for 10 minutes in 4° C and stored in 1.5 ml tubes at - 80° C until the assay (no longer than 6 months). The following secretory factors and markers of inflammation will be determined: angiogenin (ANG), • insulin-like growth factor-1 (IGF-1), • Growth Differentiation Factor 15 (GDF15), • nerve growth factor (NGF), • Amyloid Precursor Protein- α (sAPPα), • angiopoietin (ANGP1), • brain-derived neurotrophic factor (BDNF) • growth differentiation factor 15 (GDF-15), • tumor necrosis factor α (TNFα).
The general assessment of the homoeostasis Automated Hematology Analyzers
• blood morphology, • glycemia, • free iron, • activity of alanine aminotransferase (ALT), • aspartate aminotransferase (AST), • creatine kinase (CK), • lactate dehydrogenase (LDH), • concentration of insulin, • total cholesterole • creatinine,
Measurement of proteins involved in antioxidant defence using Western Blotting Techniques
Description: Qualitative detection of single proteins involved in antioxidant defence using western blotting technique. Proteins involved in antioxidant defence, and inflammation will be identified and measured: • involved in antioxidant defense (superoxide dismutase \[SOD1\], SOD2, glutathione peroxidase \[GPx\]), • inflammation (interleukin-6).
Messurment of cfDNA changes
cfDNA will be collected into cell-free DNA tubes (Roche) and isolated using Quick-cfDNA Serum \& Plasma Kit (Zymo Research) according to the manufacturer's protocol. Obtained material will be aliquoted, stored in -200C and subsequently used for further analyses. Blood samples will be collected into cell-free DNA tubes before physical exercise (in the morning, on an empty stomach), immediately after the end of trial, 5 minutes and 60 minutes after the end of exercise. Collected material will be stored in a refrigerator or on ice for cfDNA isolation
Measurement of isolated serum cytotoxic activity
The LNCaP cell line will be cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin, and incubated in a humidified atmosphere at 37°C and 5% CO2. The cytotoxic activity will be evaluated using the method described by Paredes-Gamero with some modifications. Cells will be plated onto 96-well microplates (105 cells/mL) in RPMI 1640 supplemented with 10% FBS in the absence or presence of the EEHS (25-200 μg/mL) for 24 h in a humidified atmosphere at 37°C and 5% CO2. After this period, the cells will be washed with PBS and resuspended in buffer (0.01 M HEPES, pH = 7.4, 0.14 M NaCl and 2.5 mM CaCl2). The stained cells will be fluorescently labeled and analyzed using an Accuri C6 flow cytometer and Accuri C6 software.
Body composition analyses using bioelectrical impedance analysis (BIA)
The participant's body mass and height, leg lean mass (LLM) of lower limbs will be measured by a multi-frequency bioelectrical impedance method using the body composition analyser - Inbody 720 (Biospace, Korea, Seoul). Weight and height will be combined to report BMI in kg/m\^2, and all specific analyses will give percent of muscle mass in every limb and part of body. The measured values will be served for normalisation of the peak torque outcome
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Gdansk University of Physical Education and Sport
Lead sponsor
Medical University of Gdansk
Collaborator
Kazimierz Wielki University
Collaborator
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