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
Effects of ketamine on macrophage function and platelet
aggregation
中文摘要
Ketamine 是臨床上常用的靜脈麻醉藥物，具有止痛和失憶的效果，此種中樞神
經系統的藥理作用主要是藉由對
N-methyl-D-aspartate 受體的拮抗作用。除
了在中樞神經系統的作用之外，
ketamine 對體內其他系統功能也會造成影響。
本研究的目的在探討
ketamine 於中樞神經系統之外的作用，包括：免疫功能、
凝血與血管張力調節三方面，並選擇巨噬細胞、血小板與內皮細胞進行體外實
驗，以檢測
ketamine 對其影響及可能之作用機轉。
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
tumor 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
TNF-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。
英文摘要
Ketamine (2-[2-chlorophenyl]-2-[methylamino]-cyclohexanone), a widely used intravenous anesthetic, produces dose-related unconsciousness and analgesia. N-Methyl-D-aspartate (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 attempted 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. Exposure 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
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 ability of macrophages was suppressed by ketamine. Treatment with lipopolysaccharide induced TNF- , IL-1 , and IL-6 mRNA in macrophages. Administration of ketamine alone did not influence TNF- , IL-1 , or IL-6 mRNA production. Meanwhile, cotreatment with ketamine and lipopolysaccharide significantly inhibited lipopolysaccharide-induced 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 ability, 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 formation 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 membrane 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 endothelial 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 no effect on cell viability. When the administered
concentration reached 1000 M, ketamine caused cell death after 24 h-treatment. Treatment of endothelial cells with ketamine 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 24 h decreased nitric oxide production. Endothelial NO synthase expression was reduced. This study shows that a clinically relevant concentration of ketamine (100 M) can inhibit 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 mitochondrial 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 inhibit endothelial nitric oxide
production possibly via suppression of endothelial NO synthase. In conclusion, the present in vitro results indicate that ketamine has inhibitory effects on macrophage function, platelet aggregation, and endothelial nitric oxide production. While the mechanisms responsible for these inhibitory effects require further investigation, our findings provide evidence that ketamine influences additional physiological functions beyond central nervous system. In a clinical context these findings are potentially pertinent, but more in vivo studies are needed to elucidate the functional meaning and clinical implication.
