敗血症大鼠肝臟粒線體蛋白質之變化及熱休克前處置對其之影響

Effect of preconditioning heat shock treatment in mitochondrial proteins of liver in septic rats

郭美芳(Mei-Fang Kuo)

指導教授 ： 楊瑞成

高雄醫學大學/醫學院/醫學研究所/碩士(2003年)

摘要

臨床上，敗血症造成的多器官功能失調症候群，仍是造成病人死亡的主要原因之一，近年來有越來越多的學者將此多器官功能衰竭的現象導向為粒線體功能不良所致。本實驗室先前利用盲腸結紮及穿孔手術所誘發的敗血症動物模式中，發現在肝臟及心臟中其粒線體呼 吸鏈上之酵素活性皆呈現下降情形。另外，由以往的研究及文獻也證實，熱休克反應在敗血症的大白鼠中確實具有保護作用，但其機制仍不清楚。因此本篇論文的目的為探討熱休克前處置對敗血症動物之肝臟粒線體蛋白質的變化之影響。
利用SD（Spraque-Dawley）雄性大白鼠作為實驗的動物，將其分成兩組：加熱組和非加熱組。加熱組的老鼠，利用電毯行全身性加熱 到41.5±0.5℃，維持15分鐘，以誘發熱休克蛋白大量表現。利用盲腸結紮及穿孔手術（CLP）誘發大白鼠產生敗血症。實驗性敗血症大白鼠分別在CLP後9小時（敗血症早期）和18小時（敗血症晚期）犧牲。利用雙向電泳（2D）及銀染偵測肝臟粒線體蛋白質的變化，再以液相層析儀-質譜儀分析術(Liquid Chromatography-Mass Spectrometry-Mass Spectrum；LC/MS/MS)進行特異蛋白質的測定，並利用反轉錄酶聚合酶連鎖反應（RT-PCR），偵測mRNA的量，最後利用分光光度計（Spectrophotometer）測量其蛋白質之活性。西方點墨法分析的結果顯示：熱休克處置後24小時，Hsp就有大量的合成及表現，而於加熱後42小時仍維持相當大量的表現。在雙向電泳分析的結果：從2D膠體上可偵測到約120個點（spot），其中三個點位於相同的分子量（56.4KD），但其pH值不同，編號為粒線體蛋白質1（MP 1；PI 7.0），粒線體蛋白質2（MP 2；PI 8.0）和粒線體蛋白質3（MP 3；PI 8.5）。其中粒線體蛋白質1（MP 1；PI 7.0）和粒線體蛋白質2（MP 2；PI 8.0）在敗血症早期和晚期均呈現減少現象，給予熱休克前處置後則呈現增加或不變的現象；粒線體蛋白質3（MP 3；PI 8.5）在敗血症早期和晚期均呈現增加現象，給予熱休克前處置後則呈現回復現象。在敗血症所呈現變化的蛋白質，於單獨熱休克處置42小時後並無有意義的改變。利用液相層析儀-質譜儀分析術，簡稱LC/MS/MS進行蛋白質分析，發現粒線體蛋白質1 （MP 1）、粒線體蛋白質2（MP 2）、粒線體蛋白質3（MP 3）為同一個蛋白質: 醛脫氫酶 2 (aldehyde dehydrogenase 2；ALDH 2)。接著我們利用反轉錄酶聚合酶連鎖反應（RT-PCR），偵測ALDH 2 mRNA的量，結果mRNA 的表現在敗血症及熱休克前處置後並無差異。酵素活性分析的結果則顯示：在敗血症晚期其活性明顯降低，而在加熱組敗血症晚期的活性則有明顯回復情形。
由於ALDH 2在醣質新生的過程中可幫助一些非醣類的物質轉變成醣，以供身體所需。此外在敗血症所引起的脂質過氧化反應中，所產生的一些毒性代謝產物也需要ALDH 2的存在下方能讓其轉變為無毒性的代謝產物。除此之外，ALDH 2的轉錄後修飾作用（post-translation modification）也許是一關鍵的轉變。所以我們認為在敗血症的病理變化上，ALDH 2的作用應扮演一重要之角色，在熱休克反應的保護機制上也可提供一個新的論點。

關鍵字

敗血症 ； 醛脫氫酶粒線體 ； 盲腸結紮及穿孔手術 ； 熱休克反應

並列摘要

Sepsis is regarded as a major initiator of multiple organ dysfunction syndrome that remains a leading cause of mortality in medical critical care unit. Mitochondrial dysfunction plays an important role in the cascade. Our previous studies have shown that the ATP synthesis and the mitochondrial respiratory enzyme activities were all down-regulated in the liver of experimental septic rats. In addition, heat shock response was demonstrated to the protective effect against sepsis in rats while the mechanism is still a mystery. Accordingly, the present study was designed to investigate the proteomic alternation of hepatic mitochondria during sepsis, and explore the molecular protective mechanism of preconditioning heat shock treatment.
Spraque-Dawley rats were used as the experimental animal. They were divided into two groups: non-heated and heated. Rats of heated group were whole-bodily heated to 41.5±0.5 °C for 15 min to induce the over-expression of heat shock proteins (Hsps24) hr before sepsis induction. Sepsis was induced by cecal ligation and puncture (CLP). The rats were sacrificed 9h and 18h after CLP that was defined as early and late phase of sepsis, respectively. The mitochondrial proteins of the liver were extracted and separated by two-dimensional electrophoresis with broad immobilized pH gradient strip (PH 3-10) and SDS-PAGE. The protein spots were visualized with silver stain and analyzed by Bio-2D software.
Results of Western blot and immunochemical analysis showed that Hsp72 was over-expressed maximally at 24 and sustained till 42 hours after heat shock treatment. Hsp72 was not induced in early and late sepsis, while heated septic rats all showed Hsp72 over-expression. Results of two-dimensional electrophoresis analysis showed that around 120 spots could be separated and visualized distinctly. Among them, 3 spots with same molecular weight (56.4KD) were significantly altered in septic specimens, named MP1 (PI 7.5), MP2 (PI 8.0) and MP3 (PI 8.5). MP1 and MP2 were down-regulated in sepsis while MP3 was up-regulated coincidently. Interestingly, heat shock treatment could reverse the phenomenon. Analyzed by LC/MS/MS, the 3 spots were revealed to be an identical enzyme: aldehyde dehydrogenase 2 (EC 1.2.1.3). However, RT-PCR assay of the enzyme revealed no significance in all specimens. The enzyme activity assay showed that aldehyde dehydrogenase 2 activity was down-regulated in non-heated late septic rats and reversed in heated late septic rats. But the activities between heated and non-heated early septic rats were not different significantly.
In conclusion, we suggest that post-translation modification of aldehyde dehydrogenase 2 may play a functional role in the pathogenesis of sepsis and provide a novel protective mechanism of heat shock treatment.

並列關鍵字

sepsis ； aldehyde dehydrogenase ； mitochondrial ； CLP ； heat shock reponse

參考文獻

文 獻

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