INVESTIGATION OF THE PROTECTIVE EFFECT OF BOLDIN AGAINST ROTENONE TOXICITY IN SH-SY5Y CELLS

Project no. 22429 · Status: Kapanmış · 2021

Summary

In parallel with the prolongation of human life, the incidence of neurodegenerative diseases has increased. It is estimated that 50 million people in the world suffer from neurodegenerative diseases and this figure will increase to 115 million people by 2050. Parkinson's disease, one of these diseases, is the second most common neurodegenerative disease after Alzheimer's disease, affecting more than 10 million people worldwide. It is a chronic and progressive disease that develops with dopamine deficiency in the striatum as a result of damage and loss of dopaminergic neurons located in the substantia nigra region of the brain, and progresses with tremor, bradykinesia, rigidity and postural instability. Although the etiopathogenesis of the disease has not been fully resolved, studies in the literature point to factors such as aging, genetic factors, and neurotoxicity caused by environmental toxins such as pesticides and heavy metals. The current data suggest that prevention or early treatment of PD may be the most effective therapeutic strategy. Therefore, in vivo and in vitro experimental Parkinson's models are created to elucidate the etiopathogenesis of the disease and to develop new treatment strategies. One of these models is the rotenone toxicity model. Rotenone is an organic pesticide that is often used to establish an experimental Parkinson's model in animal and cell studies. The electron transport system in mitochondria increases oxidative stress through Complex 1 inhibition. It produces Parkinson's disease-like effects in cell and animal studies.In this thesis study, toxicity was created with rotenone, an organic pesticide, in order to create an in vitro Parkinson model in human neuroblastoma cell line SH-SY5Y cells. It has been reported in the literature that various antioxidants such as flavonoids, vitamins E and C are protective against Parkinson's disease and rotenone toxicity. In our study, we aimed to investigate the neuroprotective effect of boldin, an alkaloid used to treat liver and digestive system disorders in Latin American traditional medicine, which has reported strong antioxidant and anti-inflammatory effects. In the study, increasing doses of rotenone were applied to SH-SY5Y cells for 24 hours to find the lethal dose (LD50) of rotenone. It was determined by the MTS assay that in the group exposed to 200 nM rotenone for 24 hours the cell count was reduced by approximately half that of the untreated controls. In order to determine the protective efficacy of Boldin, various doses of boldin pretreatment were applied to SH-SY5Y cell groups for 2 hours. Cell survival in groups exposed to 200 nM rotenone for 24 h after Boldin pretreatment was assessed by the MTS assay. The clonogenic assay was applied to evaluate the colony forming capacity of the cells. Hoechst staining was performed to detect apoptotic cell death and active caspase 3 was evaluated by ELISA assay.The findings obtained as a result of our study showed that rotenone is neurotoxic in SH-SY5Y cells and induces apoptotic cell death. The test results revealed that low doses of boldin have a neuroprotective effect by increasing cell survival against rotenone-induced toxicity, and this increase may be due to the antiapoptotic activity of boldin. In addition, it showed that low doses of boldin also promoted cell proliferation and colony formation potential.

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