Neurodegenerative changes occur due to contribution of many pathological processes. One of them causes increased lysosomal proteolytic activity, among them also increased activity of cysteine cathepsins, which leads to neuronal degeneration and development of neurodegenerative disorders such as Parkinson’s disease (PD). In case of PD, advanced loss of dopaminergic neurons in substantia nigra is present. Because of the involvement of cysteine cathepsins, including cathepsin X, in the development of neurodegenerative disorders, their synthetic inhibitors are seen as potential substances in the development of the PD treatment strategy. Therefore, in the master’s thesis, we concentrated on the evaluation of action of the specific synthetic reversible inhibitors of cathepsin X (Z7 and Z9) using the cell model of PD. We used neuron-like SH-SY5Y cells, whose degeneration was induced with neurotoxin 6-OHDA. We found out that inhibitors Z7 and Z9 are not toxic for SH-SY5Y cells at lower concentrations (5 and 10 µM), while small toxic influence was seen at higher concentrations. Moreover, both inhibitors Z7 and Z9 at lower concentrations did not affect a mitochondrial membrane potential of cells. In this way, the highest non-toxical concentration of both inhibitors was established, which was further used for the experiments. For the inhibitors evaluation cell model of PD was established with the use of adequate concentration of 6-OHDA, where 200 µM concentration showed an optimal toxic effect on SH-SY5Y cells. On the established cell model of neurodegeneration we further evaluated protective effects of inhibitors Z7 and Z9 at 10 µM concentration on 6-OHDA-induced toxicity of SH-SY5Y cells. We found, that pretreatment of cells with inhibitors leads to lower proportion of death and apoptotic cells as well as a milder mitochondrial membrane potential destruction was observed in comparison to 6-OHDA-treated cells. These effects were showed for both inhibitors. Additionally, both inhibitors Z7 and Z9 have been shown to reduce the increased activity of caspase-3 in 6-OHDA-stimulated SH-SY5Y cells, with the Z9 inhibitor being more effective. The experimental findings of this master’s thesis indicate the protective action of componuds Z7 and Z9 against the induced degenerative processes and loss of neuron cells. Therefore, the cathepsin X inhibitors represent a great potential for the development of new therapeutically useful agents for the treatment of neurodegenerative diseases such as PD.
|
