麻醉藥物

麻醉藥物 propofol 與 ketamine 抑制巨噬細胞功能的分子機轉研究

MOLECULAR STUDIES ON THE SUPPRESSIVE EFFECTS OF

PROPOFOL AND KETAMINE ON MACROPHAGE FUNCTION

中文摘要 手術前後期間免疫系統的調節與維持平衡是非常重要的議題。免疫系統失調會造 成嚴重的術後合併症，甚至影響病患長期的結果。手術麻醉長久以來就被認為會 影響免疫與發炎反應，無論是經由直接影響免疫活性細胞或是間接調節壓力反應 的結果。雖然有許多 in vitro 與 in vivo 研究，目前仍無定論。但是越來越多的研 究結果顯示麻醉藥物會抑制免疫發炎反應，特別是對一些免疫功能異常、加護病 房的重症病患甚至癌症病患，上述高危險群病患，很容易因為免疫功能失調而產 生 SIRS（systemic inflammatory response syndrome）、MODS（multiple organ dysfunction syndrome），甚至死亡。除了手術麻醉外，更大的挑戰是加護病房中 重症病患的鎮靜（ICU sedation），而加護病房的鎮靜藥物又幾乎是靜脈麻醉藥 物。所以靜脈麻醉藥物的免疫調控研究對上述高危險群病患是有其臨床意義的。 本研究的目的是探討臨床上常用的兩種靜脈麻醉藥物：propofol 與 ketamine 的免 疫調控作用，利用 in vitro 研究方法觀察其對巨噬細胞功能的影響與其對發炎性 細胞素（proinflammatory cytokines）基因表現調控的訊息傳遞機轉。 本研究分四個部分進行，第一部分探討 propofol 對巨噬細胞吞噬作用、氧化能 力、細胞素生成的影響；第二部分探討 propofol 對巨噬細胞驅化性移動的影響； 第三部分探討 ketamine 對 tumor necrosis factor-???n（TNF-?恁^與 Interleukin-6 （IL-6）基因表現的訊息傳導機轉；第四部分探討 propofol 對 IL-6 基因表現的訊 息傳導機轉。希望能釐清靜脈麻醉藥物的免疫調控機轉並能對高危險手術與重症 病患的照顧有所助益。

第一部分的研究結果顯示在臨床相關濃度（clinically relevant concentration）的 propofol 會抑制巨噬細胞的吞噬作用、氧化能力、發炎性細胞素的生成。其作用 機轉是作用在粒線體，影響粒線體膜電位（mitochondrial membrane potential）， 進而影響 ATP 的生成，而並非 propofol 對細胞的毒性作用。

第二部分的研究結果顯示臨床治療濃度的 propofol（50 ?嵱）會專一性的抑制粒 線體膜電位，而不是 complex I NADH 去氫酶活性，進而影響 ATP 生成與巨噬細 胞的驅化性移動。

第三部分的研究結果顯示 ketamine 在臨床治療濃度會抑制 LPS 活化之巨噬細胞 TNF-?恁 AIL-6 protein 與 mRNA 的生合成。在訊息傳導機轉的探討，我們先使 用 siRNA 的方式 knockdown TLR-4，結果會降低 TNF-?捋 PIL-6 的產生，同時給 予 ketamine 與 TLR-4 siRNA 會使 TNF-?捋 PIL-6 的產生更加減少。接下來我們 也發現 ketamine 會抑制 JNK 磷酸化，降低 c-jun translocation 到細胞核，最後還 會抑制轉錄因子 activator protein-1（AP-1）的接合能力。由以上的結果，我們可

以推論 ketamine 抑制 TNF-?捋 PIL-6 發炎基因表現是經由 TLR-4 / JNK / AP-1 的 訊息傳遞機轉。

第四部分的研究結果顯示 propofol 在臨床相關濃度會抑制 LPS 活化之巨噬細胞 IL-6 protein 與 mRNA 的生合成。在訊息傳遞機轉的探討，我們發現 propofol 會 抑制 TLR-4 mRNA 生成，減少磷酸化 JNK 與下游 c-jun translocation 到細胞核， 進而影響轉錄因子 AP-1 的結合能力。由以上結果可推論 propofol 的免疫抑制作 用是經由 TLR-4 / JNK / AP-1 的訊息傳導機轉。 綜合而言，本研究顯示在 in vitro 巨噬細胞的模型下，臨床上常使用的兩種靜脈 麻醉藥 propofol 與 ketamine，在臨床相關的濃度下都會造成巨噬細胞功能的抑 制，特別是對發炎細胞素基因調控的訊息傳導機轉都是經由 TLR-4 / JNK / AP-1 路徑。越來越多的動物及人體研究顯示，麻醉藥物本身是具有免疫調控作用。這 種免疫抑制與抗發炎反應對高危險群手術麻醉與加護病房鎮靜病患是有其臨床 意涵，而此種免疫調控作用是否會造成長期後果的影響則需要更多研究來證明。 英文摘要

Anesthesia accompanied by surgical stress may influence the immune system that is essential for maintaining the homeostasis during the perioperative period. Severe dysregulation of the immune system may cause serious postoperative complications. Anesthetic agents are suspected of impairing various aspects of the immune and inflammatory response, either directly by disturbing the functions of

immune-competent cells or indirectly by modulation of the stress response. Despite there are many related studies, this topic remains a matter of controversy. Most investigational data have been confined to in vitro studies and are therefore separated from the homeostatic regulation network. Many in vivo studies have suggested that the immunomodulatory effects of anesthesia are negligible in comparison to the processes triggered by tissue injuries. Nevertheless, in patients with pre-existing immune dysfunctions, in high risk surgical patients, in cancer patients and even critically ill patient in intensive care unit for sedation, dysregulation of immune system may cause systemic inflammatory response syndrome, multiple organ

dysfunction syndrome, and even death. The studies on immunomodulatory effects of anesthetics may have clinical implications.

The purpose of our study was to evaluate the immunomodulatory effects of two commonly used intravenous anesthetics: propofol and ketamine. We attempted to determine the effects of the two anesthetics on macrophage activities and possible signal-transducing mechanisms on cytokine genes regulation using in vitro model. In this study, we would like to explore (1) the effects of propofol on macrophage activities: phagocytosis, oxidative ability and cytokine production, (2) the effect of propofol on macrophage chemotactic migration, (3) the signal-transducing

mechanisms of ketamine in TNF-?? and IL-6 gene expression and (4)

signal-transducing mechanisms of propofol in IL-6 gene expression. We hope the results can be valuable in elucidating the immunomodulatory effects of propofol and ketamine and also can be useful in daily clinical practice.

The results of the first part of the study demonstrated that at clinically relevant concentration, propofol can suppress macrophage activities such as phagocytosis, oxidative ability and LPS-induced cytokine production. The possible mechanism may be due to decrease mitochondrial membrane potential and adenosine triphospohate biosynthesis rather than cytotoxicity effect of propofol.

The results of the second part of the study showed that, therapeutic concentration of propofol (50 ?嵱) could specifically inhibit mitochondrial membrane potential but not complex I NADH dehydrogenase activity, thus reduce adenosine triphospohate biosynthesis and migration of macrophage.

The results of the third part revealed that therapeutic concentration of ketamine could inhibit lipopolysaccharide-stimulated macrophage TNF-?? and IL-6 protein and mRNA production. To explore the signal-transducing mechanism, by using siRNA technique to knockdown TLR-4, concomitant treatment of ketamine and TLR-4 siRNA further decrease of TNF-?n?? and IL-6 production more than alone

administration of ketamine. Also we found ketamine can inhibit the downstream JNK phosphorylation, c-jun translocation to nucleus and AP-1 binding activity. We

postulated the immunomodulatory effect of propofol might be via suppression of TLR-4-mediated JNK phosphorylation and AP-1 activation.

The results of the fourth part of the study showed that at clinical relevant concentration, propofol could inhibit IL-6 protein and mRNA production in LPS-activated macrophage. Signal-transducing mechanism demonstrated that propofol can inhibit TLR-4 mRNA, phosphorylated JNK, c-jun translocation to nucleus and transcription factor AP-1 binding activity. We postulated

immunomodulatory effect of propofol might be through suppression of TLR-4-mediated JNK phosphorylation and AP-1 activation.

Collectively, the present study showed that in vitro macrophage model, clinically relevant concentrations of propofol and ketamine can suppress macrophage activities. The proposed signal transduction pathway of proinflammatory gene expression IL-6 and TNF-?? may through TLR-4-mediated JNK phosphorylation and AP-1 activation. The suppressive effects of propofol and ketamine may have clinical implications in susceptible patients. Further studies will be needed for the long-term effect of anesthetics on high risk surgical and critically ill patients.