- 學位論文
氯氨酮與白藜蘆醇對生物活性調控之機轉研究
MOLECULAR MECHANISMS OF KETAMINE- AND RESVERATROL-INDUCED MODULATION OF BIOLOGICAL ACTIVITIES
摘要
氯氨酮（ketamine）是一種靜脈麻醉藥物，1960年代以後就一直廣泛的被使用於手術中的麻醉、鎮靜以及各種疼痛的控制。由於它對於心血管系統有較穩定的作用，因此在臨床上經常被使用於血壓不穩定卻又需要手術麻醉或鎮靜的重症患者。Ketamine主要是經由肝臟的單氧酶酵素系統cytochrome P450所代謝。除了在中樞神經系統發揮作用，對重要生理產生影響，也有不少研究著重於ketamine與免疫系統的關聯性。 Ketamine必須通過血腦障壁才能影響神經細胞。血腦障壁是由一連串的腦血管內皮細胞所形成的緊密結構，而且具有選擇性的通透功能，因此能適當的保護中樞神經系統免於外來物質的攻擊。本實驗室初步的結果顯示，ketamine的確會破壞血腦障壁緊密接合的功能。其他學者的研究也顯示，ketamine會產生過量的氧化物引起中樞神經系統病生理的變化。所以我們也嘗試探討過量的氧化物對於血腦障壁的影響以及抗氧化物的保護作用。 因此，本研究分為兩部分進行：第一部分分別以HepG2細胞與Raw 264.7細胞為模型，探討ketamine對肝臟細胞代謝作用以及對於被革蘭氏陽性菌產生的內毒素脂磷壁酸活化後的巨噬細胞的影響。第二部分則分別以腦血管內皮細胞與小鼠為模型，採用中樞神經系統中常見的氧化物—氧化態低密度脂蛋白或是高脂食物，以及一種飲食中的抗氧化物—白藜蘆醇為研究素材，探討氧化態低密度脂蛋白或是高脂食物對於腦血管內皮細胞或血腦障壁產生破壞後，白藜蘆醇具有的保護作用及其機轉。 結果我們的研究發現，臨床濃度的ketamine不但會干擾肝細胞細胞支架的結構，並進而抑制肝細胞中CYP450酵素系統的表現及功能；還會透過影響TLR-2/ERK/ NFκB的信息傳遞路徑，抑制LTA活化巨噬細胞增加TNF-α和IL-6 mRNA的表現與活性氧化物的產生。而氧化態低密度脂蛋白與長期餵食高脂食物都會影響緊密接合蛋白的結構，進而破壞腦血管內皮細胞與血腦障壁的功能。但是白藜蘆醇則可以保護腦血管內皮細胞或血腦障壁，免於過量氧化物或高脂食物的傷害，並減少伴隨而來的腦神經細胞的破壞。
並列摘要
Ketamine is a neuroleptic anaesthetic agent in clinical use since the 1960’s. The mechanisms of action of ketamine on central nervous system might be an N-methyl-D-aspartate receptor antagonist, and terminated by combination of redistribution from the CNS to peripheral tissues and hepatic biotransformation by the cytochrome P-450 system. In a clinical environment, ketamine has more stable hemodynamics than barbiturates or inhaled anesthetic agents, so it is usually used in critically ill patients to induce anesthesia. In the cellular host defense system, ketamine causes a significant reduction in leucocyte activation during sepsis, while it also suppresses pro-inflammatory cytokine production in vitro. Ketamine is highly lipid soluble, so it crosses the blood brain barrier（BBB）easily. The BBB, a unique feature of the CNS, is formed by specialized cerebrovascular endothelial cells that exist in brain microvascular blood vessels. The presence of the BBB results in the nearly complete separation of the CNS from the rest of the body, and the main structures responsible for the barrier properties are tight junctions. Our preliminary data showed that ketamine might destroy the normal function of tight junctions. Previous studies also revealed that ketamine participates in the generation of oxidative stress, might play an important role in the pathophysiology of models of schizophrenia. Therefore, we attempted to evaluate the protective effects of antioxidants on oxidative stress-induced injuries of BBB. The purposes of this research were to evaluate the modulatory effects of ketamine on HepG2 cells and macrophages and the effects of oxidant / antioxidant on BBB. In the first part, we would like to explore（1）the effects of ketamine on F-actin and microtubular cytoskeletons of HepG2 cells and ketamine-caused cytoskeleton disruption on CYP3A4 expression, and（2）the effects of ketamine on TNF-α and IL-6 biosynthesis and ROS production in LTA-activated macrophages and its possible mechanisms from the viewpoint of TLR-2-mediated signal-transducing phosphorylation of ERK and the consequent translocation and transactivation of transcription factor NFκB. In the second part, we would like to explore（1）the effects of resveratrol on oxLDL-induced injuries of CECs from the viewpoints of tight junction construction and antiapoptosis, and（2）the effects of resveratrol on high-fat diet-induced disruption of the BBB and its possible mechanisms. The results of the first part of studies demonstrated that therapeutic concentrations of ketamine can disrupt F-actin and microtubular cytoskeletons possibly through suppression of intracellular calcium mobilization and cellular ATP synthesis due to downregulation of the mitochondrial membrane potential and complex I enzyme activity. Such disruption of the cytoskeleton may lead to reductions in CYP3A4 activity in HepG2 cells. On the other hand, ketamine decreased the biosyntheses of TNF-α and IL-6 in LTA-activated macrophages. A possible mechanisms is through downregulating TLR-2-mediated phosphorylation of ERK1/2 and the subsequent translocation and transactivation of NFκB. The results of the second part of studies revealed that resveratrol can protect against oxLDL-induced breakage of the BBB through preventing disruption of the tight junction structure and apoptotic insults to CECs. Further in vivo study showed that resveratrol can attenuate the high-fat diet-induced disruption of the BBB via interfering with occludin and ZO-1 tight junctions, and consequently protects against insults to brain neurons.
參考文獻
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