多菌性敗血症時肝細胞發生自體吞噬現象之探討

Autophagy occurs in hepatocytes during polymicrobial sepsis

簡薇珊(Wei-Shan Chien)

指導教授 ： 許勤

高雄醫學大學/醫學院/生理及分子醫學研究所/碩士(2007年)

https://doi.org/10.6832/KMU.2007.00082

摘要

敗血症是感染後發炎反應所導致，在重症加護病房中死亡率仍居高不下。嚴重的發炎反應會活化NFκB並產生大量的細胞激素, 如TNFα, 和氧化物質，進而導致粒線體受損和細胞能量供應不足。且氧化壓力與胞器受損都是誘發自體吞噬的原因。自體吞噬是一種在壓力的環境下胞內重新再利用營養物質，以維持能量供應恒定的生存機制。而過去相關文獻指出若抑制NFκB活性，會經由抑制mTOR而促進自體吞噬；可推測，敗血症時，NFκB之活性增加可能抑制自體吞噬。此外，已知肝臟屬於高度需能器官且在敗血症晚期會功能衰竭。故假設敗血症時自體吞噬受抑制可能導致晚期肝功能衰竭。利用大鼠進行盲腸結紮與穿刺手術，模擬腹膜炎導致敗血症的動物模式進行研究。並偵測不同時間點，肝臟中p70s6k蛋白質磷酸化(代表mTOR蛋白活性)，及自體吞噬的標誌蛋白(LC3-II)含量與聚集；利用小片段干擾核醣核酸(siRNA) 降低ATG7(LC3-II蛋白聚集之必要因子)，以確定自體吞噬在TNFα導致肝功能損傷中所扮演的角色。結果顯示: (1) 自體吞噬標誌(LC3-II)在手術後6小時達到最高峰，之後便急遽下降至18小時；(2) 肝組織中磷酸化P70s6k在手術後6小時顯著減少；(3) 自體吞噬發生在肝細胞的比例遠大於庫佛氏細胞中；(4) 利用ATG7 小片段干擾性核醣核酸減少自體吞噬現象發生，使得肝細胞在TNFα給予的情況下，細胞功能較未給予小片段干擾性核醣核酸之細胞更差。以上結果顯示，在多菌性敗血症晚期，肝細胞失去具有再回收作用的自體吞噬伴隨著肝臟衰竭。但仍需直接證據才能瞭解肝臟自體吞噬現象在敗血症時所扮演的角色。

關鍵字

自體吞噬 ； 肝細胞 ； 敗血症

並列摘要

Sepsis results in the host response to infection and remains a high mortality rate in ICU. Severe inflammation usually causes overproductions of pro-inflammatory cytokines, i.e. TNFα, and ROS that leads to mitochondrial damage and energy depletion. Oxidative stress and injured organelle are inducers of autophagy, a survival mechanism for eukaryote to recycle intracellular nutrients and maintain the intracellular energy homeostasis under stress. However, previous report indicated that the blockage of NFκB activation enhances autophagy via suppressing mTOR activity, implying NFκB activation during sepsis may suppress autophagy. Since liver is a high energy demanding organ and its’ function is failed at late stage of sepsis, hypothesized that the suppression of autophagy contributes to liver dysfunction at the late stage of sepsis. The rat model of cecal ligation and puncture (CLP) was performed to stimulate clinical peritonitis-induced sepsis and measure the time course change of phosphor-p70s6k, a substrate of mTOR that inhibits autophagy. Protein abundance of LC3-II, a marker of autophagy, and LC3 aggregation were quantified by Western blot analysis and immunohistochemistry, respectively. The siRNA was appled to knocked down the ATG7 that is essential for LC3-II aggregation to confirm the role of autophagy in TNFα-induced hepatocyte dysfunction. Our results showed that: (1) level of LC3-II significantly increases at 6 h and then declines until 18 h after CLP, (2) in liver tissue, the phosphor-p70s6k significantly declines at 6 h after CLP, (3) the ratio of LC3 aggregation in hepatocytes is significantly higher than that in kupffer cells, (4) The decreased of autopahgy process lead hepatocyte function to go worse after TNFα treatment. The present results indicate that the loss of recycle function of autophagy in hepatocytes is associated with the hepatic failure at the late stage of polymicrobial sepsis. The direct evidence needs to confirm the role of autophagy in liver during CLP induced sepsis, respectively.

並列關鍵字

Autophagy ； hepatocyte ； sepsis

參考文獻

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