延腦鼻端腹外側區粒線體呼吸鏈功能於實驗性內毒素血症之變化研究

敗血症起因於身體對抗感染或受傷害的一種過度發炎反應。代謝異常、不正常血流及在細胞層次降低氧的利用率，常是造成組織傷害及導致多種器官衰竭及死亡的原因。細胞能量的產生主要來自粒線體，因此敗血症可能造成粒線體功能異常而導致生物能量衰竭引起多種器官失去功能。
本實驗室發現動脈壓訊號頻譜功率密度低頻成份之變化與交感神經性血管運動張力有關，並且反應腦幹的整合功能。此低頻成份源於腦幹延腦鼻端腹外側區（rostral ventrolateral medulla, RVLM）之前運動交感神經元，與生物體之"生-死"過程有相關聯性。此動物模式提供實驗性內毒素血症中，RVLM連續性的神經活性變化。建立於此模式下，本論文探討在敗血症終至死亡過程中，RVLM粒線體功能的改變。此外，評估一種新型的水溶性輔酶Q10（coenzyme Q10 , CoQ10）於RVLM對抗實驗性內毒素血症造成死亡之神經保護作用。


大白鼠實驗性內毒素血症延腦鼻端腹外側區粒線體功能之異常

本實驗研究急性內毒素血症造成粒線體呼吸功能的改變。在使用propofol麻醉下的大白鼠，經靜脈注射大腸桿菌內毒（lipopolysaccharide, LPS）30 mg/kg造成漸進性的低血壓，並且在4個小時內死亡。因LPS引起的內毒素血症可分為三期：第一期為動脈壓頻譜功率密度的血管運動成份（0-0.8 Hz）降低；第二期為動脈壓頻譜功率密度的血管運動成份放大（"pro-life"期）；第三期為血管運動成份第二度降低（"pro-death"期）。粒線體酵素分析顯示在RVLM處，NADH cytochrome c reductase（Complex I+III）及cytochrome c oxidase（Complex IV）於急性內毒素血症三個時期專一酶活性均是降低。而succinate cytochrome c reductase（Complex II+III）的活性則沒有改變。


CoQ10在延腦鼻端腹外側區對抗大白鼠因實驗性內毒素血症致死之神經保護作用

CoQ10是一個位於粒線體呼吸鏈上高度可移動的電子攜帶者，並且是一個抗氧化劑。本實驗評估一種新型的水溶性CoQ10在RVLM對抗實驗性內毒素血症致死之神經保護作用。於propofol麻醉的大白鼠中，以LPS（30 mg/kg）誘發實驗性內毒素血病。先前於RVLM兩側微量注射CoQ10（1 mg或 2 mg）處理的動物，死亡率明顯降低，存活時間延長並減緩因LPS引起的低血壓。於RVLM以CoQ10微量注射先前處理亦明顯延長內毒素血症之第二期時間及增加第二期動脈壓頻譜功率密度血管運動成份之總和。因LPS引起的內毒素血症第二期及第三期，RVLM之superoxide anion生成量增加亦因給予CoQ10而減緩。

結論

在致死的急性內毒素血症中，位於RVLM處的粒線體呼吸鏈酶Complex I及Complex IV的功能發生異常。CoQ10作用於RVLM處，對抗實驗性內毒素血症造成的致死效果則呈現神經保護作用。CoQ10減少RVLM神經細胞對superoxide anion的過度生成是CoQ10神經保護作用的機轉之一。本論文研究顯示粒線體功能在敗血症的重要角色，亦提供了治療高死亡率的敗血症一個新的方向。

Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia

Sepsis is a complex pathophysiologic state resulting from an exaggerated whole-body inflammatory response to infection or injury. Metabolic disturbances, abnormal regulation of blood flow and diminished utilization of oxygen at the cellular level may account for tissue damage and lead to multiple organ failure and death. As the primary site of cellular energy generation is the mitochondrion, it presents itself as an important target for the septic cascade. In this regard, the notion that bioenergetic failure due to mitochondrial dysfunction contributes to organ failure during sepsis has received attention.
We established the low frequency fluctuations in the systemic arterial pressure signals are related to the sympathetic neurogenic vasomotor tone, and reflect the functional integrity of the brain stem. Their origin is subsequently traced to the premotor sympathetic neurons at the rostral ventrolateral medulla (RVLM), whose neuronal activity is intimately related to the “life-and-death” process. Based on a rat model of experimental endotoxemia that provides continuous information on changes in neuronal activity in the RVLM, the present study was undertaken to evaluate whether changes in mitochondrial respiratory functions are associated with death arising from sepsis. We also evaluated the efficacy of a new water-soluble coenzyme Q10 (CoQ10, ubiquinone) formula in the protection against fatality during endotoxemia by microinjection into bilateral RVLM.

Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia in the Rat

We investigated the functional changes in mitochondrial respiratory chain at the RVLM in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. Experiments were carried out in adult male Sprague-Dawley rats that were maintained under propofol anesthesia. Intravenous administration of E. coli lipopolysaccharide (LPS; 30 mg/kg) induced progressive hypotension, with death ensued within 4 hours. The sequence of cardiovascular events during this LPS-induced endotoxemia can be divided into a reduction (Phase I), followed by an augmentation (Phase II; “pro-life” phase) and a secondary decrease (Phase III; “pro-death” phase) in the power density of the vasomotor components (0-0.8 Hz) of systemic arterial pressure (SAP) signals. Enzyme assay revealed significant decrease of the activity of NADH cytochrome c reductase (Complex I+III) and cytochrome c oxidase (Complex IV) in the RVLM during all 3 phases of endotoxemia. On the other hand, the activity of succinate cytochrome c reductase (Complex II+III) remained unaltered.

Neuroprotective Effects of Coenzyme Q10 at Rostral ventrolateral Medulla Against Fatality During Experimental Endotoxemia in the Rat

CoQ10 is a highly mobile electron carrier in the mitochondrial respiratory chain that also acts as an antioxidant. We evaluated the neuroprotective efficacy of CoQ10 against fatality in an experimental model of endotoxemia, using a novel water-soluble formulation of this quinone derivative. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of E. coli LPS (30 mg/kg) induced experimental endotoxemia. Pretreatment by microinjection bilaterally of CoQ10 (1 or 2 mg) into RVLM significantly diminished mortality, prolonged survival time, and reduced the slope or magnitude of the LPS-induced hypotension. CoQ10 pretreatment also significantly prolonged the duration of Phase II endotoxemia and augmented the total power density of the vasomotor components of SAP signals in Phase II endotoxemia. The increase in superoxide anion production induced by LPS at the RVLM during Phases II and III endotoxemia was also significantly blunted.

Conclusion
The present study revealed that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM is closely associated with fatal endotoxemia. CoQ10 provides neuroprotection against fatality during endotoxemia by acting on the RVLM. We further found that a reduction in superoxide anion produced during endotoxemia at the RVLM may be one of the mechanisms that underlie the elicited neuroprotection of CoQ10. These findings therefore open a new direction for future development of therapeutic strategy in this critical, complicated and highly fatal condition known as sepsis.

目 錄

中文摘要 ………………………………………………………… I
英文摘要 ………………………………………………………… V
縮寫表 ………………………………………………………… XII
第一章 緒論與文獻回顧 …………………………………………… 1
1-1 粒線體及呼吸鏈 …………………………………………… 2
1-1-1 粒線體的結構及功能 ………………………………… 2
1-1-2 吸呼鏈及氧化磷酸化反應 …………………………… 3
1-1-3 粒線體的基因組 ……………………………………… 5
1-2 粒線體與疾病的關係 ……………………………………… 6
1-2-1 粒線體疾病 …………………………………………… 6
1-2-2 粒線體與細胞死亡 …………………………………… 12
1-2-3 粒線體，氧化反應物的產生與疾病的關係 ………… 12
1-3 敗血症與粒線體功能的變化 ……………………………… 15
1-3-1 敗血症及相關疾病 …………………………………… 15
1-3-2 敗血症時粒線體呼吸功能異常的証據—細胞病理性 缺氧（cytopathic hypoxia）……………………………… 18
1-3-3 敗血症時粒線體顯微結構的變化 …………………… 19
1-3-4 敗血症時粒線體呼吸功能的變化 …………………… 19
1-3-5 敗血症引起粒線體功能異常的可能機轉 …………… 23
1-4 輔酶Q10（CoQ10）…………………………………………… 26
1-4-1 CoQ10在粒線體電子傳遞鏈的角色…………………… 28
1-4-2 CoQ10的抗氧化作用及其他功能……………………… 28
1-4-3 CoQ10在臨床上的使用………………………………… 29
1-4-4 CoQ10的藥物動力學…………………………………… 30
1-4-5 CoQ10的神經保護作用………………………………… 30
1-4-6 CoQ10在敗血症的角色………………………………… 31
1-5 動脈壓頻譜分析 …………………………………………… 33
第二章 研究動機與目的 …………………………………………… 36
2-1 研究動機 …………………………………………………… 37
2-2 研究目的 …………………………………………………… 39
第三章 實驗材料與方法 …………………………………………… 40
3-1 動物處理 …………………………………………………… 41
3-2 動脈壓及頻譜分析 ………………………………………… 41
3-3 實驗性內毒素血症 ………………………………………… 42
3-5 由大白鼠RVLM分離粒線體 ……………………………… 42
3-6 粒線體呼吸鏈酶之分析 …………………………………… 45
3-6-1 分析NCCR (Complex I+III) ………………………… 45
3-6-2 分析SCCR (Complex II+III) ………………………… 47
3-6-3 分析CCO (Complex IV) ……………………………… 47
3-7 測量粒線體懸浮液的蛋白質濃度 ………………………… 49
3-8 水溶性CoQ10的備製 ……………………………………… 49
3-9 於RVLM微量注射水溶性CoQ10 ………………………… 49
3-10 腦幹組織切片……………………………………………… 50
3-11 測量RVLM之superoxide生成量………………………… 50
3-12 實驗資料分析……………………………………………… 52
第四章 於大白鼠實驗性內毒素血症延腦鼻端腹外側區粒線體 之功能異常 ………………………………………………… 53
4-1 簡介 ………………………………………………………… 54
4-2 實驗步驟 …………………………………………………… 56
4-3 結果 ………………………………………………………… 57
4-4 討論 ………………………………………………………… 64
第五章 Coenzyme Q10在延腦鼻端腹外側區對抗大白鼠因實驗性 內毒素血症致死之神經保護作用 ………………………… 68
5-1 簡介 ………………………………………………………… 69
5-2 實驗步驟 …………………………………………………… 71
5-3 結果 ………………………………………………………… 71
5-4 討論 ………………………………………………………… 85
第六章 綜合討論 …………………………………………………… 89
6-1 結論 ………………………………………………………… 90
6-2 本論文之意義與特色 ……………………………………… 91
6-3 本論文的應用價值 ………………………………………… 93
6-4 本論文的限制 ……………………………………………… 94
6-5 未來展望 …………………………………………………… 95
參考文獻 ………………………………………………………………97
附錄……………………………………………………………………123

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