Analysis of Cardiolipin and Monolysocardiolipin Fluctuation during Inflammation and Apoptosis by LC-MS.

• Main Authors: CHANG, WAN-HSIN, 張菀芯
• Other Authors: HSU, YUAN-HAO
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/95993597033447409586

碩士 === 東海大學 === 化學系 === 104 === Macrophage is responsible for the innate immunity, involving the phagocytosis of pathogens and the cytokine secretion to induce immune responses. During macrophage activation, less unsaturated cardiolipin (CL) species increase and highly unsaturated CL species decrease. We previously have confirmed that the supplementation of omega-3 fatty acid, including docosahexaenoic (DHA) and eicosapentaenoic acid (EPA), and omega-6 fatty acid (arachidonic acid, AA) alters fatty acid composition of CL. Here we dedicated in finding the changes of CL and monolysocardiolipin (MLCL) species induced by fatty acid release during inflammation. We activated macrophage cell line RAW264.7 via KdO2-Lipid A (KLA) as our inflammatory cellular model. The extracted mitochondrial cardiolipin species, including CL and MLCL were analyzed by LC-MS. The results revealed that after KLA stimulation, less unsaturated CL species increase and high unsaturated CL species decrease. After supplementation of AA、EPA and DHA, with or without activation, the total amount of CL decreases and MLCL increases. Moreover, the percentage of long chain species elevates and short chain species reduces in both CL and MLCL. We further examined whether the changes of CL species distribution caused by omega-3 and omega-6 fatty acids are iPLA2-dependent. Co-treatment of (S)-BEL and (R)-BEL, inhibitors of iPLA2β and γ, to RAW cell resulted in the increase of CL containing three or more oleic acid (18:1) within the C70 and C72 groups. Our results strongly indicate that iPLA2s play important roles in CL remodeling in macrophages, and suggest the high specificity of iPLA2β to CL containing oleic acid (18:1). Extrinsic and intrinsic pathways are two underlying mechanisms of apoptosis. The extrinsic pathway induced by the death signals, which stimulate their receptors embedded within cell membrane to cause cell death via caspase cascade, such as TNF-α. On the other hand, the intrinsic pathway is initiated by organelle damages from environmental stress, which induces release of cytochrome c from mitochondrial inner membrane, leading to apoptosis. Therefore, cytochrome c release is considered as an important indicator of mitochondria-mediated apoptotic pathway. It has been known that cardiolipin interacts with cytochrome c, so we hypothesize that the changes of cardiolipin composition can be an indicator of cell apoptosis. In this study, we initiated human fibrosarcoma cells (HT-1080) apoptosis by H2O2 and co-treatment of TNF-α and cycloheximide (CHX), and further analyzed the total amount and composition of cardiolipin by LC-MS. The result showed that after TNF-α treatment the total amount of cardiolipin does not change, but the percentage of short chain species increase and long chain species decrease. However, co-treatment of TNF-α and CHX, an apoptosis enhancer, resulted in no obvious changes in total amount of cardiolipin and it composition. Treatment of H2O2 did not change the total amount and the composition of cardiolipin. Therefore, the results suggest that TNF-α/CHX and H2O2 induced cell apoptosis did not disturb the structure of inner mitochondrial membrane.