Toxoplasma gondii ile enfekte insan trophoblastik hücrelerinin (BEWO) toll-like reseptör yolağına nitrofurantoinin etkisi

EDA SİVCAN

Toxoplasma gondii ile enfekte insan trophoblastik hücrelerinin (BEWO) toll-like reseptör yolağına nitrofurantoinin etkisi

Thesis Type: Doctorate

Institution Of The Thesis: Erciyes University, Veteriner Fakültesi, Klinik Öncesi Bilimler, Turkey

Approval Date: 2019

Thesis Language: Turkish

Student: EDA SİVCAN

Supervisor: Abdullah İnci

Open Archive Collection: AVESIS Open Access Collection

Abstract:

Toxoplasmosis is a common parasitic disease caused by the obligatory intracellular protozoan parasite Toxoplasma gondii. The disease causes serious clinics from fetus to adulthood. The aim of this study was to investigate the effect of nitrofurantoin (NF) on the signaling pathway of TLR (Toll-Like Receptor) in the BeWo cell line, a human trophoblastic choriocarcinoma cell infected with T. gondii. BeWo cells were infected with tachyzoites in a 1: 1 ratio. NF was added to the cells forming the experimental groups to determine the concentration to be used. Giemsa staining method, trypan blue staining method and MTS (3-(4,5-dimetiltiyazol-2-yl)-5-(3-karboksimetoksifenil)-2-(4-sülfofenil)-2H-tetrazolyum) test were applied. After MTS measurement, IC50 (maximal inhibitory concentration) and EC50 (Half maximal effective concentration refers to the concentration of a drug) values were calculated using GraphPad Prism 8.0 statistical program. The appropriate drug concentration was found to be 1.25 µM (micromolar). To determine the effect of NF on the TLR pathway, samples were collected at three different times; 24 h, 48 h and 72 h. After the experiment, cDNAs were synthesized using RNA isolated from the samples taken at 24, 48 and 72 hours and Real-Time PCR (RT-qPCR) analyses were performed. Proportional change in gene expression was calculated using GAPD (Glyceraldehyde-3-phosphate dehydrogenase) and B2M (Beta-2-microglobulin) reference genes. According to this; due to the decrease in expression level in MAPK1 (Mitogen-activated protein kinase 1), SPP1 (Secreted phosphoprotein 1), TLR6 (Toll-like receptor 6) and TLR8 (Toll-like receptor 8) genes, the most effective time zone of the drug was found to be 48th hour. In addition, no significant difference was observed in the change levels of the parasitic load in the three experimental groups after 24 hours (p = 0.086), but after 48 (p = 0.015) and 72 (p = 0.049) hours, there was a significant difference in the parasitic load changes in the three experimental groups. As a result, it was shown that NF inhibited T. gondii infection by which genes in the TLR pathway. Furthermore, the knowledge of these molecules, which are stimulated by the effective dosage of NF used in the fight against toxoplasmosis, have shown that new preparations with fewer side effects and a wide range can be developed as an alternative method of treatment of the disease. Keywords: BeWo cell line, Nitrofurantoin, Toll-like receptor, Toxoplasma gondii.