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  • 學位論文

靜脈麻醉藥物PROPOFOL對脂磷壁酸活化巨噬細胞生成一氧化氮之抑制作用研究

SUPPRESSIVE EFFECTS OF INTRAVENOUS ANESTHETIC PROPOFOL ON NITRIC OXIDE SYNTHESIS IN LIPOTEICHOIC ACID-ACTIVATED MACROPHAGES SUPPRESSIVE EFFECTS OF INTRAVENOUS ANESTHETIC PROPOFOL ON NITRIC OXIDE SYNTHESIS IN LIPOTEICHOIC ACID-ACTIVATED MACROPHAGES

指導教授 : 陳瑞明 陳大樑
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摘要


Propofol是一種被廣泛使用的靜脈麻醉藥物,由於其具有短效且起始作用迅速的特性,因而常被用於全身麻醉之誘導與維持以及加護病房病患的鎮靜安眠。在加護病房中,嚴重的敗血症以及敗血性休克是最主要的致死原因。Nitric oxide (NO)是許多重要生理功能所必須的,但是過量的NO則被認為是敗血症中導致組織傷害的重要因子。本實驗的主要目的是探討propofol對受到脂磷壁酸(lipoteichoic acid; LTA)活化之巨噬細胞產生NO的影響與其作用機轉。 將巨噬細胞以25、50及75 ?M的propofol或1、5、10、20、50及75 ?g/ml的LTA處理後發現,細胞的存活率與對照組相比並無明顯差異。將細胞以100 μM的propofol、10 μg/ml的LTA處理,或以100 μM的propofol與10 μg/ml的LTA同時處理巨噬細胞,會造成明顯的細胞毒性。接著以不同濃度的LTA刺激巨噬細胞,結果發現,NO的產生與LTA的濃度成正比。再將LTA與不同濃度的propofol同時處理細胞,發現propofol可以抑制由LTA所誘導的NO產生,而且抑制的效果與propofol的濃度成正比。為了探討propofol對NO生成的抑制機轉,所以接下來觀察iNOS 蛋白與mRNA的表現。免疫轉印分析的結果顯示,propofol可以抑制因LTA誘導所造成的iNOS蛋白增加,而反轉錄?連鎖反應分析的結果顯示,propofol可以減低LTA刺激造成的iNOS mRNA上升。為瞭解propofol抑制LTA誘導導致iNOS表現的機轉,接著觀察轉錄因子nuclear factor-κB (NF-κB)的變化。免疫轉印分析的結果顯示,propofol可以降低LTA誘導的NF-κB活化,而且propofol對於dominant negative mutant inhibitor κB所造成NF-κB轉位至細胞核的抑制作用有加成的效果。進一步探討轉錄因子NF-κB上游inhibitor κB kinase與extracellular signal-regulated kinase1/2的改變,結果發現,propofol可以抑制LTA刺激產生的inhibitor κB kinase與extracellular signal-regulated kinase1/2活化。以上結果顯示,臨床濃度的propofol可以抑制巨噬細胞因LTA誘導所產生的NO,此抑制作用與propofol降低iNOS蛋白及mRNA的表現有關。而對於iNOS表現的抑制作用,與propofol降低轉錄因子NF-κB的活化,以及propofol抑制NF-κB上游inhibitor κB kinase及extracellular signal-regulated kinase1/2的磷酸化有關。

並列摘要


Propofol, an intravenous anesthetic agent, is widely used for induction and maintenance of general anesthesia and sedation in the intensive care units because of its rapid onset and short acting. Severe sepsis and septic shock are the most important causes of death in intensive care units. Although nitric oxide (NO) is essential for several physiologic functions, excessive production of NO is believed to contribute to tissue damage in septic shock. The purposes of this study were to find out if propofol would affect NO synthesis in lipoteichoic acid (LTA)-activated macrophages and its possible mechanism. Exposure of RAW264.7 macrophages to propofol at 25, 50, and 75 ?M or LTA at 1, 5, 10, 20, 50, and 75 ?g/ml did not affect cell viability. However, 100 ?M propofol, 100 ?g/ml LTA, and a combination of 100 ?M propofol and 10 ?g/ml LTA led to significant cell death. Exposure of macrophages to LTA at different concentrations increased the levels of nitric oxide in a concentration-dependent manner. While macrophages were treated with a combination of propofol and LTA, propofol could concentration-dependently decrease the NO production in LTA-activated macrophages. To explore the inhibition of NO production by propofol, the expression of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA were examined. Immunoblotting analysis showed that propofol could decrease LTA-induced iNOS protein. Analysis by a reverse transcriptase-polymerase chain reaction revealed that the LTA-induced iNOS mRNA expression was inhibited by propofol. To clarify the mechanism by which propofol inhibited LTA-induced iNOS expression, transcription factor nuclear factor κB (NF-κB) was investigated. Immunoblotting analysis showed that propofol could inhibit LTA-induced NF-κB activation. Furthermore, the upstream inhibitor κB kinase (IKK) and extracellular signal-regulated kinase1/2 (ERK1/2) were examined. The results revealed that propofol could suppress LTA-induced NO production. These suppressive effects were associated with down-regulation of iNOS protein and mRNA by propofol. The inhibition of NF-κB activation as well as phosphorylation of IKK and ERK1/2 led to suppression of iNOS by propofol.

並列關鍵字

lipoteichoic acid NF-κB nitric oxide macrophages iNOS

參考文獻


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