The effects of the dietary lipid Conjugated Linoleic Acid (CLA) and its 9:11 isomer on the expression of soluble receptors for advanced glycation end-products (sRAGE) in THP-1 Monocytic cells
University of Wales Institute Cardiff
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This study aimed to investigate the hypothesis that the dietary lipid Conjugated Linoleic Acid (CLA) and its 9:11 isomer are capable of inducing the expression of the soluble variant of the Receptor for Advance Glycation End-Products (RAGE) known as sRAGE in monocytic cells. RAGE is an important cell-surface receptor for various ligands, in particularly AGE. The interaction of AGE and RAGE is believed to transduce pro-inflammatory intracellular signals that lead to the vascular cell damage found in common complications of Type 2 Diabetes Mellitus (T2DM). However the formation of sRAGE, brought about by the removal of the transmembrane domain, forms secreted non-membrane bound variants of RAGE, which act as decoys to which the AGE ligands interact with, which limits AGE interaction with RAGE, therefore suppressing RAGE signalling, which leads to the vascular impairment of cells such as monocytes. CLA and its 9:11 isomer have been implicated in the reduction of vascular cell damage. Therefore evaluating their effect on sRAGE will enable further understanding of the mechanisms by which they reduce inflammation. This investigation involved treating THP-1 monocytic cells with CLA and 9:11 over time periods of 1, 4, 24, 48 and 72 hours at two different concentrations (20µM and 100µM). A set of standards were set up to produce a standard curve and equation to determine the concentration of the proteins extracted from the tissue culture cells. Once the protein concentrations were determined, the Western Immunoblotting technique was employed in order to detect protein levels of sRAGE. The concentrations of the proteins were successfully determined using the equation obtained from the standard curve, which showed positive correlation between the absorbance and concentration of the standard solutions, proving its reliability. However, no acceptable blots were acquired in this investigation, as no signal was produced by any of the protein samples or even the MagiMark protein ladder used. This was most likely due to an error in the transfer of the proteins and the protein ladder from the gel to the membrane. Possible errors include too low a current, incorrect gel-composition, improper transfer buffer components or insufficient transfer time. Therefore in conclusion, no results were obtained for the detection of sRAGE protein levels in cell lines treated with CLA and its 9:11 isomer. This meant that the aim of investigating how three different variables (lipid type, concentration and time) affect the production of sRAGE in monocytic cells could not be achieved. As a result, continued investigation into this particular field of study is required.
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