動脈粥狀硬化是在動脈血管壁中一種慢性的發炎反應，有很多不同的細胞參與其中，包含內皮細胞，平滑肌細胞和巨噬細胞。在動脈粥狀硬化斑塊中，發現氧化型低密度脂蛋白 (ox-LDL) 表現量很高，而ox-LDL會引發細胞產生很多初發炎性的cytokines和生長因子。過去研究顯示細胞在受到氧化刺激下會釋放的分子Cyclophilin A (CypA)也可能是一種初發炎性的cytokine。因此本研究中我們想證實，ox-LDL是否會使巨噬細胞釋放出CypA。利用西方點墨法 (Western Blot) 分析胞外蛋白CypA的表現量，我們發現當巨噬細胞處理ox-LDL時，分泌型CypA表現量會隨著時間增加而增加。我們也觀察胞內蛋白的表現量，發現磷酸化p38和磷酸化Erk的蛋白表現量都有增加。雖然我們並沒有發現MMP-9的活化情況有明顯的改變，但是西方點墨法結果顯示MMP-9有增加的現象。再者，利用外加CypA結果顯示，巨噬細胞在分泌型CypA的刺激下，MMP-9的活化情況雖然沒有明顯的增加，但磷酸化p38和磷酸化Erk的蛋白表現量卻有增加。另一方面，CD147是分泌型CypA在細胞膜上的接受器，而我們利用了免疫螢光染色去分析CD147的表現，在外加CypA或ox-LDL的情況下，在細胞膜上CD147的表現量會累積增加。總而言之，我們的結果指出，在ox-LDL的處理下，由單核球 (monocyte) 分化的巨噬細胞會分泌出CypA並且活化MAPK pathway。而分泌型CypA會促使CD147的表現量在細胞膜上聚集，並且活化ERK pathway。此外，藉由免疫組織染色(immunohistochemistry)實驗顯示，在餵食高膽固醇飲食(模擬動脈粥狀硬化的動物模型)兔子腎臟組織中，CypA會堆積在腎絲球細胞周圍，因此我們使用腎絲球腎隔細胞 (MES-13) 在處理H2O2 或高血糖所引發的氧化壓力之下，分析分泌型CypA的表現量。發現當MES-13處理H2O2 或高葡萄糖後分泌型CypA的表現量都會隨著改變。或許分泌型CypA除了在動脈粥狀硬化疾病發展早期中扮演著重要的角色，而此分泌型CypA也是腎臟發生早期發炎性反應的重要因子。 Atherosclerosis is a chronic inflammatory response in the walls of arteries. Different types of cells are involved in the atherosclerosis progression, including endothelial cells, smooth muscle cells and macrophages. Oxidized low-density lipoproteins (ox-LDLs), with high concentrations in atherosclerotic plaques, induce the production of numerous proinflammatory cytokines and growth factors. Cyclophilin A (CypA) is a secreted oxidative stress-induced factor that promotes inflammation. In the present study, we examined whether ox-LDL induces macrophages to secrete CypA. Western blot analysis showed that secreted CypA increases with time in the cultured macrophages treated with ox-LDL. Furthermore both phospho-p38 and phospho-Erk levels are increased in the cells treated with ox-LDL. In addition, we observed an increased MMP-9 expression by Western blot analysis. Next, we assessed whether recombinant CypA stimulates and regulates MMP-9 in the cultured macrophages. Under CypA stimulation, phospho-p38 and phospho-Erk levels are increased upon CypA treatment. Because CD147 is a signaling receptor for extracellular CypA, we performed immunofluorescence analysis to study the expression of CD147. The data showed an increased accumulation of CD147 on cell membrane upon the treatment of CypA or ox-LDL. Taken together, our results indicate that monocyte-derived macrophages secrete CypA and activate MAPK pathway under ox-LDL treatment. Extracellular addition of CypA in the cultured macrophages leads to an increased accumulation of CD147 on cell membrane and ERK activation. Furthermore, as evidenced by immunohistochemical staining, the accumulation of CypA was observed among glomerular cells in the kidney tissues of the rabbits treated with high cholesterol diet. The cultured mesangial cells were treated with oxidative stress induced by H2O2 or high glucose. Western blot analysis showed that secreted CypA expression increases with time in the cultured mesangial cells treated with H2O2 or high glucose. This thesis provides evidence that secreted CypA acts as an inflammatory mediator that may be involved in the pathogenesis of atherosclerosis. Moreover, CypA may also act as an inflammatory factor in the initial stage of nephropathy.
