麻醉藥物 ketamine 對巨噬細胞功能與血小板凝集的藥理作用之研究

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第一部份的研究使用小鼠巨噬細胞株 Raw 264.7 ，評估巨噬細胞的四項功能：驅化性移動、細胞吞噬作用、氧化能力以及發炎性細胞素的生 成。以 10 或 100 microM 的 ketamine 處理巨噬細胞，經過 1 、 6 或 24 小時後，均不會影響細胞的存活率，也不會影響乳酸去氫酶的釋出量。

若以 1000 microM 的 ketamine 處理巨噬細胞，則會造成乳酸去氫酶的釋出及細胞死亡。 10 、 100 或 1000 microM 的 ketamine 不會影響巨噬 細胞的驅化性移動。 ketamine 會抑制巨噬細胞的吞噬作用與氧化能力。以脂多醣活化巨噬細胞時，巨噬細胞會被誘發生成發炎性細胞素 tu mor necrosis factor-alpha （ TNF-alpha ）、 interleukin-1 （ IL-1 beta ）和 interleukin-6 （ IL-6 ），而 ketamine 則會抑制脂多醣的誘發作用，

使巨噬細胞內 TNF-alpha 、 IL-1 和 IL-6 mRNA 表現減少。 ketamine 降低巨噬細胞粒線體的膜電位，卻不影響 complex I NADH 去氫酶的 活性。因此，臨床相關濃度的 ketamine （ 100 microM ）會抑制巨噬細胞的三種功能，包括細胞吞噬作用、氧化能力以及發炎性細胞素 TN F-alpha 、 IL-1 beta 、和 IL-6 的生成，其機轉可能是藉由降低巨噬細胞粒線體的膜電位，而非 ketamine 直接的細胞毒性。

第二部份的研究使用人類血小板，來評估血小板的凝集反應及其機轉。不論在人類富含血小板血漿或血小板懸浮液中， ketamine （ 100-350 microM ）均會抑制血小板凝集反應，且抑制效果與 ketamine 濃度成正相關。對於 collagen 活化的血小板， ketamine 會抑制 phosphoinositide 的裂解反應以及細胞內鈣離子的移動，也會抑制 thromboxane A2 的生成。此外， ketamine 增加血小板細胞膜上嵌入的 diphenylhexatriene 螢 光強度，表示血小板細胞膜的流動性會被降低。 ketamine 抑制 P47 蛋白質的磷酸化，因此 protein kinase C 也會受到 ketamine 的抑制。因此

， ketamine 具有抑制血小板凝集反應的作用，其機轉可能藉由血小板細胞膜流動性的改變，因而影響細胞膜上的 phospholipase C 活性，導 致 phosphoinositide 裂解及 P47 蛋白質磷酸化反應受到抑制，進而降低細胞內鈣離子移動與 thromboxane A2 生成，最終使得血小板凝集被抑 制。第三部份的研究使用人類臍靜脈內皮細胞，來評估內皮細胞生成一氧化氮的能力。以 1 、 10 或 100 microM 的 ketamine 處理內皮細胞，經 過 1 、 6 或 24 小時後，均不會影響細胞的存活率。但若以 1000 microM 的 ketamine 處理 24 小時後，則會造成細胞的死亡。以 100 microM 的 ketamine 處理內皮細胞，經過 1 、 6 或 24 小時後，一氧化氮的釋出量會隨著 ketamine 作用時間而減少，內皮細胞一氧化氮合成酶蛋白質 的表現量也會隨著 ketamine 作用時間而減少。因此，臨床相關濃度的 ketamine （ 100 microM ）會抑制內皮細胞生成一氧化氮，其機轉可能 是藉由細胞內抑制內皮細胞一氧化氮合成酶的表現，而非 ketamine 直接的細胞毒性。

綜合以上三個部份的體外細胞實驗結果， ketamine 可能藉由降低粒線體的膜電位，造成巨噬細胞功能的抑制，包括細胞吞噬作用、氧化能 力、以及發炎性細胞素 TNF-alpha 、 IL-1 beta 、和 IL-6 的生成； ketamine 也可能藉由改變血小板細胞膜的流動性，導致 phosphoinositide 裂 解及 P47 蛋白質磷酸化反應受到影響，進而降低細胞內鈣離子移動與 thromboxane A2 生成，造成血小板凝集被抑制。此外， ketamine 也會 抑制內皮細胞一氧化氮合成酶的表現，使得內皮細胞一氧化氮的生成減少。至於 ketamine 對生物體內免疫功能、凝血、與血管張力調節的 實際影響與臨床相關意義，則是值得繼續進一步探討的研究主題。

麻醉藥物 ketamine 對巨噬細胞功能與血小板凝集 的藥理作用之研究

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Ketamine (2-[2-chlorophenyl]-2-[methylamino]-cyclohexanone), a widely used intravenous anesthetic, produces dose-related unconsciousness and analgesia. N-Methyl-D-a spartate (NMDA) receptor antagonism accounts for most of the neuropharmacological effects of the compound. Besides central nervous system actions, ketamine might alter other physiological functions. The purpose of our studies is to evaluate the influences of ketamine on immune function, hemostasis, and vascular tone regulation. We attempt ed to determine the effects of ketamine on macrophage, platelet, and endothelial cell functions and the possible mechanisms using in vitro experimental model.

In the first part of the studies, we evaluated the effect of ketamine on macrophage function and its possible mechanism using mouse macrophage-like Raw 264.7 cells. Expos ure of macrophages to 10 and 100 M ketamine, which correspond to 0.1- and 1-times the clinically relevant concentration, for 1, 6, and 24 h had no effects on cell viability  or lactate dehydrogenase release. When the administered concentration reached 1000 M, ketamine caused a release of lactate dehydrogenase and cell death. Ketamine, at 10  , 100 M, and 1000 M, did not affect the chemotactic activity of macrophages. Treatment of macrophages with ketamine reduced phagocytic activities. The oxidative abilit   y of macrophages was suppressed by ketamine. Treatment with lipopolysaccharide induced TNF- , IL-1 , and IL-6 mRNA in macrophages. Administration of ketamine alon   e did not influence TNF- , IL-1 , or IL-6 mRNA production. Meanwhile, cotreatment with ketamine and lipopolysaccharide significantly inhibited lipopolysaccharide-induc   ed TNF- , IL-1 , and IL-6 mRNA levels. Exposure to ketamine led to a decrease in the mitochondrial membrane potential. However, the activity of mitochondrial complex I   NADH dehydrogenase was not affected by ketamie. This study shows that at a clinically relevant concentration, 100 M ketamine can suppress phagocytosis, oxidative abilit  y, and inflammatory cytokine production of macrophage possibly via reduction of the mitochondrial membrane potential instead of direct cellular toxicity.

In the second part of the studies, we tested the effect of ketamine on human platelet aggregation and examined the possible mechanism. Ketamine concentration-dependently (100-350 M) inhibited platelet aggregation both in washed human platelet suspensions and platelet-rich plasma stimulated by agonists. Ketamine inhibited phosphoinositide  breakdown and intracellular calcium ion mobilization in human platelets stimulated by collagen. Ketamine (200 and 350 M) significantly inhibited thromboxane A formati   on stimulated by collagen. Moreover, ketamine (200 and 350 M) increased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation  of a platelet protein of Mr 47,000 (P47), a substrate of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (100 nM). This phosphorylation was markedly inhibited by ketamine (350 M). These results indicate that the antiplatelet activity of ketamine may be involved in the following pathways. Ketamine may change platelet m  embrane fluidity, causing activation of phospholipase C and subsequent inhibition of phosphoinositide breakdown and phosphorylation of P47, thereby leading to inhibition of intracellular calcium ion mobilization and thromboxane A formation, ultimately resulting in inhibition of platelet aggregation. 

In the third part of the studies, we evaluated the effects of ketamine on endothelial nitric oxide production and its possible mechanism using human umbilical vein endothelia l cells. Exposure of endothelial cells to 1, 10 and 100 M ketamine, which correspond to 0.01-, 0.1- and 1-times the clinically relevant concentration, for 1, 6, and 24 h had n  o effect on cell viability. When the administered concentration reached 1000 M, ketamine caused cell death after 24 h-treatment. Treatment of endothelial cells with ketami  ne time-dependently reduced nitric oxide (NO) production and endothelial nitric oxide synthase expression. Exposure of endothelial cells to 100 M ketamine for 1, 6, and 2  4 h decreased nitric oxide production. Endothelial NO synthase expression was reduced. This study shows that a clinically relevant concentration of ketamine (100 M) can i  nhibit endothelial nitric oxide production possibly via suppression of endothelial NO synthase instead of direct cellular toxicity.

In summary, ketamine can suppress macrophage function of phagocytosis, its oxidative ability, and inflammatory cytokine production possibly via reduction of the mitochon drial membrane potential. Ketamine also changes platelet membrane fluidity, with subsequent reduction of phosphoinositide breakdown and phosphorylation of P47, thereby leading to decreased intracellular calcium ion mobilization and thromboxane A formation, ultimately resulting in inhibition of platelet aggregation. Besides, ketamine can in  hibit endothelial nitric oxide production possibly via suppression of endothelial NO synthase. In conclusion, the present in vitro results indicate that ketamine has inhibitory e ffects on macrophage function, platelet aggregation, and endothelial nitric oxide production. While the mechanisms responsible for these inhibitory effects require further inv estigation, our findings provide evidence that ketamine influences additional physiological functions beyond central nervous system. In a clinical context these findings are p otentially pertinent, but more in vivo studies are needed to elucidate the functional meaning and clinical implication.

Effects of ketamine on macrophage function and platelet aggregation