肽聚醣誘導人類臍靜脈內皮細胞凋亡之機轉   全文下載

Molecular Mechanisms in Peptidoglycan-Induced Human Umbilical Vascular Endothelial Cell Apoptosis

革蘭氏陽性菌細胞壁成分?聚醣（Pgn）被認為是革蘭氏陽性菌誘導敗血症相關併發症的主要分子，然而?聚醣誘導血管內皮層功能喪失的致病機轉仍未完全釐清。此外aSK1蛋白被報導在許多不同刺激誘導細胞凋亡的過程中扮演重要角色，因此本研究主要是想探討革蘭氏陽性菌細胞壁成分?聚醣是否可經由活化細胞內aSK1蛋白而導致人類臍靜脈血管內皮細胞凋亡。我們發現?聚醣可隨著濃度增加而降低細胞存活率，流式細胞儀分析更說明了?聚醣可誘導血管內皮細胞凋亡，?聚醣被證實可活化細胞內aSK1蛋白及其下游訊息分子p38maPK蛋白，同時?聚醣也會增加細胞內凋亡蛋白Bax的表現。另一方面，由於?聚醣會結合至細胞上的tlR2受體，因此我們也使用細胞tlR2受體的致效劑Pam3CSK4，實驗發現Pam3CSK4和?聚醣的作用相似，可誘導人類臍靜脈血管內皮細胞aSK1和p38maPK的磷酸化以及細胞凋亡。由這些結果推測革蘭氏陽性菌細胞壁成分?聚醣可能經由活化細胞內aSK1-p38maPK訊息傳遞路徑，誘導凋亡蛋白Bax的表現，最終使得細胞凋亡。藉由了解?聚醣誘導血管內皮細胞功能喪失的作用機轉，如果可以使用藥物或是其他方式阻斷細胞內aSK1-p38maPK訊息傳遞路徑，將有助於發展臨床上治療因革蘭氏陽性菌感染所導致的血管相關疾病的有效治療策略。

Peptidoglycan (PGN), a component of the outer membrane of Gram-positive bacteria has been implicated in the pathological process of sepsis. However, the molecular mechanism of PGN-induced vascular endothelium dysfunction has not been fully elucidated. Apoptosis signal regulating kinase 1 (ASK1) has been recently reported to play a crucial role in cell apoptosis under various cellular stresses. The purpose of this study was thus to investigate whether PGN-activated ASK1 results in cell apoptosis in human umbilical vascular endothelial cells (HUVECs). PGN was shown to cause a decrease in cell viability in a concentration-dependent manner. Flow cytometric analysis demonstrated that PGN increased the percentage of apoptotic cells. PGN induced ASK1 activation was accompanied by the increased phosphorylation of p38MAPK, a major downstream signaling molecule of ASK1. In addition, PGN was shown to increase apoptotic protein, Bax, expression in HUVECs. Inhibitor of p38MAPK signaling abrogated the PGN-increased DNA fragmentation and Bax expression, suggesting functional crosstalk. The toll-like receptor 2 (TLR2) agonist, Pam3CSK4, was also shown to induce ASK1 activation and p38MAPK phosphorylation, and subsequent cell apoptosis in HUVECs. Our data suggest that ASK1-p38MAPK cascade activation, followed by Bax expression, contributes to PGN-induced cell apoptosis.