Vrednotenje reverzibilnih sinteznih zaviralcev katepsina X na 6-hidroksidopaminskem celičnem modelu Parkinsonove bolezni

Virant, Monika (author), Pišlar, Anja (mentor)

Nevrodegenerativne spremembe nastanejo zaradi seštevka številnih patoloških procesov. Eden izmed njih je povečana aktivnost lizosomalnega proteolitskega sistema, med njimi tudi cisteinskih katepsinov, kar vodi do propada nevronov in razvoja nevrodegenerativnih bolezni kot je Parkinsonova bolezen (PB). Pri PB pride do napredne izgube dopaminergičnih nevronov v črnem jedru. Zaradi vpletenosti cisteinskih katepsinov, pri razvoju nevrodegenerativnih bolezni, med njimi tudi katepsina X, so njihovi sintezni zaviralci potencialne zdravilne učinkovine pri načrtovanju strategij zdravljenja PB. V magistrski nalogi smo se zato osredotočili na ovrednotenje delovanja specifičnih sinteznih reverzibilnih zaviralcev katepsina X, in sicer Z7 in Z9 na celičnem modelu PB. Pri tem smo uporabili nevronske celice SH-SY5Y, katerih propad smo spodbudili z uporabo nevrotoksina 6-OHDA. Ugotovili smo, da zaviralca Z7 in Z9 nista toksična za celice SH-SY5Y pri nižjih koncentracijah (5 in 10 µM), medtem ko pri višjih izkazujeta manjši toksični vpliv. Zaviralca v nižjih koncentracijah tudi ne izkazujeta vpliva na porušenje mitohondrijskega membranskega potencial. Tako smo določili najvišjo še ne toksično koncentracijo obeh zaviralcev, ki smo jo uporabili za nadaljnje poskuse. S pomočjo ustrezne koncentracije 6-OHDA smo postavili celični model PB, pri čemer se je koncentracija 200 µM izkazala kot optimalna za postavitev modela. Na postavljenem celičnem modelu nevrodegeneracije smo nadalje ovrednotili zaščitno delovanje zaviralcev Z7 in Z9 za celice SH-SY5Y pri koncentraciji 10 µM po izpostavitvi le-teh nevrotoksinu 6-OHDA. Ugotovili smo, da s pred-tretiranjem celic z zaviralcema pride do manjšega števila mrtvih in apoptotičnih celic ter do blažjega porušenja mitohondrijskega membranskega potenciala v primerjavi s celicami, ki smo jih stimulirali samo s 6-OHDA. Tovrstne zaščitne učinke sta izkazovala oba zaviralca. Hkrati smo pokazali, da zaviralca Z7 in Z9 zmanjšata povišano aktivnost kaspaze-3 v s 6-OHDA-stimuliranih celicah SH-SY5Y, pri čemer se je zaviralec Z9 izkazal za učinkovitejšega. Izsledki eksperimentalnega dela tako nakazujejo na zaščitno delovanje zaviralcev Z7 in Z9 na 6-OHDA celični model procesa nevrodegeneracije in propada nevronskih celic. Zaviralca zato predstavljata potencial za razvoj novih terapevtsko uporabnejših učinkovin za zdravljenje nevrodegenerativnih bolezni, kot je PB.

• Language: Slovenian
• Keywords: Parkinsonova bolezen, 6-hidroksidopamin, Nevroblastomske celice SH-SY5Y, Katepsin X, Zaviralci katepsina X
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-108336
• Publication date in RUL: 28.06.2019

Secondary language

• Language: English
• Title: Evaluation of Reversible Synthetic Cathepsin X Inhibitors in the 6-OHDA cell Model of Parkinson's Disease

Abstract

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.

• Keywords: Parkinson's disease, 6-Hydroxydopamine, Neuroblastoma cells SH-SY5Y, Cathepsin X, Cathepsin X inhibitors