Evaluation of the effect of Silybum marianum (L.) seed extract on PI3K/akt/mTOR pathway and dna repair in NRK-52E cell line.

Ceran F, Evaluation of the Effect of Silybum Marianum (L.) Seed Extract on PI3K/Akt/mTOR Pathway and DNA Repair in NRK-52E Cell Line. Health Sciences Institute, Department of Biochemistry (Veterinary Medicine), M.Sc. Thesis, Van, 2025. It is well known that oxidative stress leads to damage in intracellular pathways and genetic systems. Silymarin and its derivatives, found in Silybum marianum extract (SME), have been reported to exert protective effects against cellular damage, particularly protecting the liver and other tissues, and enhancing protein expression. This thesis aims to investigate the role of SME in preventing H₂O₂-induced genotoxic damage and its impact on the PI3K/Akt/mTOR pathway. Since the kidney is one of the organs most affected by intracellular metabolic events, a rat kidney cell line was selected as the study model. The experimental groups were established as Control, Serum Starvation (SS), SME, H₂O₂ (HP), and SME+HP. The concentrations of H₂O₂ and SME to be applied to the cells were determined using the MTT assay. Genomic damage was assessed using the comet assay, and gene expression levels were determined by RT-qPCR analysis. The proliferative concentration of SME was determined to be 25 μg/ml, and the cytotoxic concentration (IC25) of H₂O₂ was identified as 30 μM. After treatment with SME and HP according to their respective groups, total mRNA was isolated from the cells at the end of 8 hours. Expression levels of the obtained mRNAs were then evaluated. In the HP-treated group, a significant increase in genomic damage was observed compared to the control group (p<0.05). However, in the SME+HP group, genomic damage was significantly reduced compared to the HP group (p<0.05). The mRNA expression levels of TP53 and PARP-1 genes were found to be decreased in the HP group compared to the control, while their levels were higher in the SME+HP group compared to the HP group. In the HP group, the expression levels of target genes within the PI3K/Akt/mTOR pathway were significantly reduced compared to the control group (p<0.05), whereas in the SME+HP group, the expression levels of these genes approached those of the control. Based on these findings, it was determined that H₂O₂ at a cytotoxic concentration induces genomic damage and reduces the expression levels of PI3K/Akt/mTOR pathway components as well as the repair genes TP53 and PARP-1. In conclusion, Silybum marianum extract has been shown to reduce oxidative stress-induced genotoxic damage under in vitro conditions and to modulate adverse alterations in the PI3K/Akt/mTOR pathway, indicating its potential as a protective agent in cellular defense. Further in vivo and clinical phase studies are necessary to clearly support these findings.