Clinical Research Directory

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,

Ischemic Preconditioning - Perspectives of Use Vivio/in Vitro Studies

Both in sports and medicine, methods that can significantly contribute to improving the ability of tissues to perform their functions are constantly being searched. One of those methods that is increasingly used also in sports is remote ischemic preconditioning (RIPC). Mostly, this procedure involves repeated brief cycles of limb ischemia/reperfusion. This method is often referred as ischemic blood reperfusion and helps to increase the tolerance of treated tissues to the occurrence of possible ischemic episodes in the future. Numerous studies have shown that RIPC induces changes that lead to increased resistance to hypoxia and other stressors in organs (brain, heart, liver). In addition, it has been proven that induction of arterial occlusion in the area of selected limbs before performing physical exercises can affect the improvement of their function, and thus can translate into sports results. In addition, an adaptation of such fibres to damage is mostly associated with the secretion of many factors that influence body function. That's why we conclude that it may affect protein kinases (such like c-Jun N-terminal kinases (JNKs) or serine-threonine kinase = protein kinase B (AKTs)) whose main role is regulation of the activity of a wide spectrum of substrates, influencing cells proliferation, apoptosis, responses to cellular stress and inflammatory process in normal and cancer cells. Concluding that presented kinases activity is associated with cells differentiation and RIPC and physical activity may affect them and the inflammation process that may lead to cytotoxic activity against cancer cells (especially if the effects are combined together). The beneficial increase of anti-tumour activity of the blood serum against pathological isolated tumour cells of prostate cancer (cell lines - LNCaP and PC- 3) was confirmed in our pilot studies. We observed an increase in the anti-cancer properties of serum taken from people that attended RIPC training and performed physical activity. However, the exact mechanism and associated changes in the proteome of blood serum people attending in the RIPC training have not yet been determined. This knowledge would allow us to determine the exact mechanisms of the reperfusion/reocclusion training on the human body and its beneficial activity. Considering that the skeletal muscle is an organ capable of synthesis and release of a number of proteins, cytokines and low molecular weight compounds, especially during physical activity, it should be assumed that intermittent muscle ischemic episodes will lead to increased release of factors that will increase the resistance of muscles and other tissues to stress. At the same time, it was noticed that under conditions of muscular stress induced changes in iron metabolism occur. The binding of free iron through the ferritin protein at the cell level leads to its greater resistance to stressors. In the prism of the above considerations, the results of our preliminary studies showing that the upper limb RIPC procedures cause changes in iron metabolism in white blood cells and may suggest that the procedure of RIPC leads to changes that allow storing iron in a safe form in as ferritin. At the same time, with the increasing interest in iron metabolism, the role and function of amyloid precursor protein (APP) and hepcidin are increasing. It has been proven that APP is a protein that works with ferroportin, thus taking part in iron export from a cell. Moreover, it has been shown that the post-translational modification of APP leads to the formation of amyloid α (determines positive changes) and amyloid β (negative changes). Because there are some indications that sAPPα may be modified by iron changes and associated with cfDNA changes, which substantially increase during i.e. tissue damage, we would like to explore those correlations more deeply. The same decrease in the APP protein level will lead to the inhibition of iron export from the cell and an increase of its concentration in the cell. The nature of such changes in iron metabolism should be considered as adaptive to the ischemic stress on which muscle is exposed during the RIPC procedure. The increase in ferritin in the cell leads to a decrease in the concentration of free iron and thus a reduction in iron-dependent ROS formation. This project will have an impact on the development of the current state of knowledge of the mechanisms of biochemical response to the specific tissue-affecting method in form of remote ischemic preconditioning and will allow determining the role of sAPPα and Cathepsin C and other trophic factors and changes in iron metabolism in this process, taking into account the role of hepcidin and vitamin D. Moreover, the present project may contribute to the determination of the role of presented procedures on cells proliferation, as an example of anti-tumour proprieties, and changes of human serum proteomes.