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  • 學位論文

PMC與YC-1抑制革蘭氏陽性菌細胞壁成分lipoteichoic acid引發鼠類巨噬細胞產生一氧化氮合成的作用機轉探討

Mechanisms of PMC and YC-1 Involved in the Inhibition of Lipoteichoic Acid-Induced Nitric Oxide Production in Murine Macrophage RAW 264.7 Cells

指導教授 : 許準榕 博士
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摘要


中文摘要 在各種發炎疾病的狀態中,一氧化氮 (nitric oxide; NO) 是一個重要的調節及作用分子。當組織在發炎時,大量的NO會藉由誘導型一氧化氮合成酵素 (inducible nitric oxide synthase; iNOS) 產生。特別是內毒素所引起之敗血性休克時,iNOS產生過量的NO被認為是導致低血壓,低收縮反應性血管及組織氧化性傷害的主要原因之一。 本篇論文主要探討並比較不同作用方式之PMC與YC-1對於革蘭氏陽性菌細胞壁成分lipoteichoic acid (LTA) 在巨噬細胞RAW 264.7產生過量nitric oxide (NO) 的影響,以進一步探討這些藥物在這其中所牽涉到的抑制機轉。在巨噬細胞RAW 264.7實驗中,我們發現LTA以濃度相關的方式刺激RAW 264.7釋放Nitric Oxide,其中以20 mg/ ml可達次高原之刺激程度,在培養液中可測得之NO含量約增加為未加藥組的3倍。當以不同濃度的PMC處理RAW 264.7細胞後,會明顯而有意義地隨劑量增加的而減少LTA (20 mg/ ml) 所誘發產生的NO, 且其抑制50 % 反應之濃度 (IC50) 為47.8 ± 4.9 mM。而以不同濃度的YC-1處理RAW 264.7細胞後,也會明顯地有意義且以隨劑量增加的方式減少LTA (20 mg/ ml) 誘發產生的NO, 且其抑制50 % 反應之濃度 (IC50) 為4.3 ± 1.3 mM。由此可知,YC-1之抑制效果約為PMC之10倍,尤其YC-1濃度在10mM下,可幾乎完全抑制NO之產生。由細胞存活實驗得知PMC與YC-1的抑制作用,並不是藉由細胞毒性所造成的。除此之外,分別投予PMC與YC-1 30分鐘後,再以LTA處理24小時,也會抑制iNOS protein及iNOS mRNA的表現量。實驗更進一步發現PMC與YC-1也會影響IkB-a的降解作用,且其抑制效果YC-1較PMC強,特別的是兩者抑制屬MAPK成員的JNK/ SAPK蛋白質活化之磷酸化反應。但YC-1濃度在10mM下,僅部分抑制。這些結果暗示著LTA引起iNOS表現之機轉主要仍以NF-kB之路徑為主。 綜合以上的發現,我們推測PMC與YC-1為其抑制LTA誘發巨噬細胞RAW 264.7釋放NO的作用機轉可能均可透過減少JNK的活化,與IkB-a的降解作用,進而抑制iNOS mRNA表現,最後導致iNOS protein減少,進而減少NO的累積。這個結果提供了PMC與YC-1抑制巨噬細胞iNOS之作用機轉。因此未來可能利用PMC與YC-1抑制iNOS的作用來治療敗血症引發的心血管系統發炎性傷害。

並列摘要


英文摘要 Nitric oxide (NO) is an important regulator and effector molecule in various inflammatory disease states. High output of NO during inflammation is generated by the inducible isoform of nitric oxide synthase (iNOS). Overproduction of NO by iNOS is thought to be the principal factor of hypotension and hyporeactivity to vasoconstrictor agents observed in endotoxic shock. The present study was to investigate the effect of PMC and YC-1, on the induction of iNOS formation by lipoteichoic acid (LTA) in cultured murine RAW 264.7 cells, a macrophage-like cell line. According to our study, in murine RAW 264.7 cells cultures, LTA could dose-dependently induce NO production. LTA (20 mg/ml) synergistically induced a 3-fold production of NO on murine RAW 264.7 cells for 24 hrs incubation. PMC and YC-1 caused a significant and concentration-dependent inhibition on the production of NO upon stimulation by LTA (20 mg/ml) in murine RAW 264.7 cells. In addition, murine RAW 264.7 cells pretreated with PMC and YC-1 by stimulation of LTA- caused a concentration-dependent reduction both in iNOS mRNA and protein expression. At the same dose, PMC and YC-1 did not significantly reduce the cell viability. Furthermore, we found that PMC and YC-1 inhibit IkB-a degradation. In addition, PMC and YC-1 significantly inhibited JNK/SAPK phosphorylation. Therefore, based on the above observations, we suggested that PMC and YC-1 diminished LTA-induced NO production in murine RAW 264.7 cells by a mechanism involving inhibition of MAPK activation, followed by the inhibition of iNOS mRNA, thereby leading to the inhibition of iNOS protein and NO production. These results suggest a possible role of PMC and YC-1 in managing septic inflammation of cardiovascular system through inhibition of iNOS induction.

參考文獻


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