Sigara Dumanıyla Uyarılan Nazal Epitel Hücrelerde Oksidatif Stres Cevabında Görev Alan SEPP1,NCF2, SFTPD, CCL5 ve PTGS1 Genlerinin Metilasyon Profili Ve Kurkumin Uyarımının Bu Profile Etkisi

Abstract

Smoking is one of the most common habits all over the world. Although it has been known for many years that smoking is harmful to health, more than 7 million people in the world die from diseases caused by smoking every year. Thousands of chemicals in cigarette smoke have carcinogenic and toxic effects and cause damage to many tissues and organs of the body, especially the respiratory system. Tobacco smoke triggers the emergence of many life-threatening diseases such as cancer, cardiovascular diseases, reproductive system disorders, respiratory system problems. As a result of exposure to cigarette smoke, oxidative stress occurs in cells exposed to chemicals in this smoke. In addition, epigenetic changes occur in these cells due to the deterioration of their molecular mechanisms, and alterations in normal gene expressions occur with mechanisms such as methylation. Gene expression alterations in terms of upregulation and downregulation is observed in the expression of SEPP1, NCF2, SFTPD, CCL5 and PTGS1 genes, which are involved in the oxidative stress mechanism. Such alterations are observed in healthy cells. Although there are studies in the literature showing that oxidative stress-induced epigenetic changes can be prevented and reversed with antioxidant compounds such as curcumin, no study has been found showing whether oxidative stress-induced changes in gene expression profiles of the aforementioned five genes can be reversed by curcumin treatment. In this thesis, changes in the expression of SEPP1, NCF2, SFTPD, CCL5 and PTGS1 genes as a result of oxidative stress in nasal epithelial cells exposed to cigarette smoke are studied to determine to what extent it is possible to reverse these changes with curcumin treatment. In the thesis study, viability tests were performed to determine the cell cytotoxicity as a result of stimulation of the RPMI-2650 cell line with curcumin and cigarette smoke. EtBr/Calcein AM staining, MTT, LDH and ROS analyzes were performed to detect viability. Expression analysis of five selected genes was performed by qPCR test on DNA obtained by mRNA isolation from cells and cDNA conversion. The epigenetic effect of curcumin on the total methylation profile was investigated by examining the total methylation profiles of the cells separately in curcumin and CSC stimulation. Within the scope of the studies, bisulfite conversion analysis was also performed and it was investigated whether the C-T nucleotide conversion resulting from CSC stimulation could be normalized by curcumin stimulation. As a result of exposure to cigarette smoke, there is a significant increase in the total methylation profiles of nasal epithelial cells compared to the control group, but this increase can be prevented and returned to a healthy level with curcumin treatment. According to the results obtained in the thesis study, both curcumin and CSC have a lethal effect at high concentrations, and curcumin should be used in concentration ranges of more than 5 μM and 50-100 μg/μL with CSC. Concentrations that are higher than the specified values for both stimuli create a lethal effect. Another finding obtained in the study is that oxidant stress and epigenetic changes in nasal epithelial cells caused by cigarette smoke can be reversed by curcumin treatment. As a result of the experiments examining the total methylation profile, a significant increase is observed in the total methylation profiles of nasal epithelial cells as a result of exposure to cigarette smoke, compared to the control group, but this increase can be prevented by curcumin treatment and returned to a healthy level. Another important finding of the thesis study is that while the expression of SEPP1, SFTPD and PTGS1 genes, which are involved in the prevention of intracellular oxidant stress, is decreased by cigarette smoke, curcumin treatment has an inhibitory effect. Similarly, while NCF2 and CCL5 genes, which are involved in the development of intracellular oxidant stress, increase in cigarette smoke expression, curcumin decreases the increased expression of these genes due to cigarette smoke. Therefore, curcumin has a regulatory role in the intracellular oxidant mechanism, and it is a compound that can show both upregulating and downregulating effects in order to bring the expressions of genes whose expression changes in the positive or negative direction as a result of CSC stimulation to normal levels.