Atopik Dermatit Hücre Kültür Modelinde Sert Suyun Kallikrein İlişkili Peptidazlar ve Oksidatif Stres Üzerine Etkisinin İncelenmesi

Abstract

Atopic dermatitis (AD) is the most common inflammatory skin disease affecting people of all ages, although it is more common in children. AD, which is characterized by skin dryness, itching and redness, is defined by skin barrier dysfunction and overactivation of the immune system. Type-2 immune response mechanism is frequently observed in the pathogenesis of AD. In addition to factors such as smoking and air pollution associated with the disease, exposure to chalky water has been found to affect the severity of AD by disrupting the integrity of the skin barrier. With its high calcium and magnesium ions, it is more alkaline (pH>8.5) than normal water and increases the physiological pH level of the skin (pH:4.5-5.3). While kallikrein-related peptidases (KLK), one of the protease groups in the skin, play a role in the shedding of aging skin in a healthy skin, at high pH levels, as a result of their increased proteolytic activity, they increase the permeability of the skin and make it vulnerable to allergens. It has been found that KLK-5 and -7, whose activity increases due to pH change, cause inflammation through the receptors TLR-2 or PAR-2 located on the epithelial cell surface of the skin. It is known that KLKs released out of the cell activate the anti-microbial peptide LL-37 and cause an increase in reactive oxygen species (ROS) in inflammatory cells. Within the scope of the thesis, the effects of pH and ion exchange with calcareous water on KLK-5, KLK-7 and LL-37 were investigated at gene (real-time PCR) and protein (ELISA) levels in differentiated keratinocytes in both AD and non-AD states. In keratinocytes, TLR-2 and PAR-2 blockers were used to investigate which receptor KLKs are more effective on epithelial cells. With the keratinocyte-eosinophil co-culture model, the role of KLKs and LL-37 released from epithelial cells stimulated with lime water in ROS production in eosinophils was investigated. Within the scope of the thesis, the effects of pH and ion exchange with lime water on KLK-5 and -7 and LL-37 were investigated at gene (real-time PCR) and protein (ELISA) levels in differentiated keratinocytes in both AD (AD(+)) and non-AD (AD(-)) states. In keratinocytes, TLR-2 and PAR-2 blockers were used to examine the receptor(s) through which KLKs act on epithelial cells. With the keratinocyte-eosinophil co-culture model, the role of KLKs and LL-37 released from the epithelial cell stimulated with lime water and the role of LL-37 in ROS production in eosinophils was investigated by real-time PCR assay. The level of KLK-5 produced from HaCaT cells as a result of stimulation of HaCaT cells both alone and after co-culturing with Eol-1 cell line was determined by ELISA method and KLK-7 and LL-37 gene expressions were determined by real-time PCR assay. As a result of ELISA and real-time PCR experiments, KLK-5 level increased in 12 h and 24 h stimulation with 180 and 200 mg/L CaCO3, especially in AD(-) conditions, and this increase was reversed or prevented by the presence of C29 and SsnB blockers. In addition to these results, KLK-5 levels were variable in AD(+) conditions, decreasing in some conditions with CaCO3 stimulation, but increasing in both CaCO3 concentrations with C29 and SsnB blockers. KLK-7 and LL-37 gene expressions were decreased by CaCO3 in the 12 h AD(-) stimulation condition, but C29 blocker reversed this effect and significantly increased gene expressions. In contrast, in AD(+) condition, KLK-7 expression increased with 200 mg/L CaCO3, decreased with C29, but increased again with SsnB. LL-37 expression increased in all conditions. In the 24 h AD(-) experimental setup, KLK-7 expression increased in all conditions. LL-37 expression also increased with CaCO3 and C29. However, in AD(+) conditions, KLK-7 expression increased 32-fold with 200 mg/L CaCO3 and this increase was attenuated by the addition of blockers to the medium. In the experiment in which ROS gene profiles were examined with RNAs isolated from Eol-1 cell line, the expression of genes such as SOD1 and ApoE, which are also used as biomarkers in AD disease, increased as a result of exposure to 180 mg/L CaCO3, while the effect of calcium carbonate was reversed by the presence of TLR-2 receptor blockers. Despite this result, the expression of the remaining oxidative stress genes was increased by the presence of SsnB blocker in both 12 and 24 hours of stimulation, independent of the AD-model. As a result of all the experiments performed, an increase in both gene (real-time PCR) and protein (ELISA) levels of KLK-5, KLK-7 and LL-37 was observed in the HaCaT cell line stimulated with CaCO3 at 2 different concentrations at 12 and 24 hours under certain conditions, independent of AD status. In addition to CaCO3 stimulation, the presence of inhibitors used as TLR-2 receptor blockers in the environment increased both the expression and protein levels of the related genes. These results obtained with TLR-2 receptor blockers suggest that the effect of CaCO3 on HaCaT cells may go through another receptor instead of the TLR-2 receptor, and the receptor blockers used may affect different cellular pathways in HaCaT cells and cause KLK-5, KLK-7 and LL-37 to be higher in gene and protein levels.