探討兩種人類麻醉用藥戊巴比妥和異丙酚對於發炎反應之影響

Abstract

第一部分 菌血症是重症病患最主要的致死原因之一，而戊巴妥是常利用在實驗動物麻醉所使用的麻醉藥物。利用清醒鼠實驗技術以及脂多醣 (LPS) 建立清醒鼠內毒素模型，並發現戊巴妥能夠降低該動物模型中發炎細胞激素甲型腫瘤壞死因子之分泌。實驗結果顯示，戊巴妥能夠降低在脂多醣刺激下甲型腫瘤壞死因子(TNF-□) 的蛋白質以及mRNA的表現量；其作用機制是透過抑制轉錄因子 NF-□B, AP-1以及胞內蛋白 p38 MAPK的活性進而導致降低甲型腫瘤壞死因子的表現。除此之外，由於甲型腫瘤壞死因子會造成細胞組織器官的壞死，因此我們進行活體內毒性測試，我們發現在戊巴妥治療組中, 老鼠血清中胺酸轉胺脢 (alanine aminotransferase, ALT)、天冬氨酸轉氨酶 (aspartate aminotransferase, AST)、乳酸脫氫脢 (lactic dehydrogenase, LDH)、肌酸肌脢 (creatine kinase, CK)、血清尿素氮(serum urea nitrogen)、澱粉酵素 (amylase) 的含量都較內毒素組明顯降低，顯示戊巴比妥能夠降低內毒素所造成之多器官傷害。由體外實驗發現戊巴比妥能夠在施予磺酸去鐵胺 (deferoxamine mesylate, DFO) 所造成的缺氧環境下降低細胞凋亡的發生率。以上結果顯示戊巴比妥不但能夠降低脂多醣所造成的發炎反應更能夠保護細胞免於直接或間接由甲型腫瘤壞死因子所引起的細胞凋亡。以上結果除了讓我們更了解戊巴比妥抗發炎的作用機制外，也提醒我們有許多在戊巴妥麻醉模式下所進行的藥物測試所觀察到的抗發炎反應結果有可能是由戊巴妥所協同作用引起的。 第二部分 以異丙酚進行麻醉或鎮靜之醫療行為時，往往伴隨著免疫細胞如巨噬細胞或自然殺手細胞之活性遭到抑制，然而已報導的相關機制並無提及是否與調控型細胞激素乙型轉型生長因子有關。為了更進一步探討臨床使用之異丙酚是否會對病人體內之乙型轉型生長因子造成變動，我們將外傷病患分為有接受或沒有接受 異丙酚治療，並檢測這些病患血清中乙型轉型生長因子的濃度。我們首次發現相較於沒有接受異丙酚的病患，在有接受異丙酚治療的病患血清中，無論是總量還是已活化的乙型轉型生長因子都明顯較高。此外我們利用細胞實驗探討異丙酚究竟是促進哪一類的細胞分泌乙型轉型生長因子。在分別針對人類血液周邊單核細胞、單核球細胞株、T 淋巴球細胞株以及人類臍帶靜脈內皮細胞投與臨床用量之異丙酚並分析其乙型轉型生長因子分泌後顯示，異丙酚會增加人類血液周邊單核細胞、單核球細胞株、T 淋巴球細胞株之培養液中活化態乙型轉型生長因子之含量。不同於免疫類細胞，異丙酚反而不影響人類臍帶靜脈內皮細胞培養液中活化態乙型轉型生長因子之表現，但卻促使該細胞分泌更多的非活態之乙型轉型生長因子。並且當我們將以異丙酚前處理過的內皮細胞培養液與單核球細胞共同培養後，相較於控制組，單核細胞的吞噬活性被抑制將近百分之二十。最後，利用乙型轉型生長因子訊息傳遞途徑的阻斷劑SB431542，我們證實了乙型轉型生長因子主導了有接受異丙酚治療的病人血清中能抑制單核細胞吞噬活性的相關機制。我們的發現有助於避免在臨床上使用異丙酚所造成的如免疫抑制等負作用。

Part I Sepsis is the leading cause of death for intensive care patients. Lipopolysaccharide (LPS) administration to animals under anesthesia is a strategy for the study of uncontrolled release of proinflammatory cytokines. Anesthetics have been indicated that they can specially affect immune responses, such as the inflammatory response. Pentobarbital is an anesthetic used mainly in animal studies. Thus, the effect of pentobarbital on tumor necrosis factor-alpha release was determined. The results revealed that pentobarbital suppressed the expression of TNF-alpha mRNA and its proteins, which may result from the decrease in the activities of nuclear factor-kappaB and activator protein 1 and the reduction of the expression of p38 mitogen-activated protein kinase by pentobarbital. After the inhibitory activity of the pentobarbital for TNF-aalpha□release was proven in vivo, the cytotoxic effects of LPS were examined in vivo with or without pentobarbital treatments. In vivo results indicated that plasma levels of alanine aminotransferase, aspartate aminotransferase, lactic dehydrogenase, creatine kinase, serum urea nitrogen, and amylase decreased dramatically in the anesthetic group with pentobarbital administration. Finally, the effect of pentobarbital on TNF-alpha□related cell death was monitored in vitro, and the results indicated that pentobarbital could directly enhance the viabilities of cells under the treatment of TNF-alpha and protected cells from apoptosis induced by deferoxamine mesylate-induced hypoxia. These results suggest that pentobarbital significantly influences the LPS-induced inflammatory responses and protects cells from death directly and indirectly induced by TNF-alpha. The information provides a perspective to re-evaluate the results of the experiments in which animals were anesthetized with pentobarbital. The anti-inflammatory effects of the drugs may have been caused by the synergistic effect of pentobarbital. Part II Propofol anesthesia or sedations downregulate the functions of many immuno-competent cells such as macrophage and neutrophils in vivo. However, the effects of propofol on regulatory cytokine TGF-beta1 secretion in vivo are unknown. To investigate the clinical use whether affect the concentration of TGF-beta1 in human, the human sera were obtained from the trauma patients treated with or without propofol and the levels of the total and active TGF-beta1 expression was measured. Besides, the effects of propofol on TGF-beta1 expression in human peripheral blood mononuclear cells (PBMC), vein endothelial cells (HUVECs), lymphocytes (Jurkat) and monocytes (THP-1) were also tested. Moreover, these sera were also tested in regards to their effects on monocytes endocytosis activity with or without TGF-beta1 pathway inhibitor SB431542. Our results revealed that propofol would raise the total amount and active form of TGF-beta1 in propofol-received patient sera (n=14) compared to non-propofol- received patient sera (n=10) within 24 h after surgical operations. Furthermore, propofol induced latent form TGF-beta1 secretions in HUVEC cells (human umbilical vein endothelial cells) and enhanced TGF-beta1 activation in THP-1 and JURKAT cells within clinical dosages (6.5□g/ml) in vitro. Besides, the sera from propofol-received patients would suppress the macrophage activity ex vivo (65.93% ± 5.36%, SE) and could be abrogated by TGF-beta1 pathway inhibitor SB431542 (113% ± 9.48%, SE). These results implied that clinical treatments of propofol could trigger endothelial cells to express latent TGF-beta1 which would be converted into the active form through monocytes or lymphocytes activities. This finding was conducive to predict the potential effects of propofol when used in medical operations during anesthesia.