| 摘要: | 背景：外傷出血性休克常造成早期死亡，晚期原因則為器官衰竭。儘管過去許多的臨床與基礎之研究，仍然無法完全明瞭其機轉。外傷出血性休克造成組織及細胞缺氧，誘發組織缺氧及再灌流傷害，產生過氧化物自由基，將引起組織進一步傷害及發炎反應。各類休克常導致肝臟衰竭。Cobb等嘗試於小白鼠之敗血模式中，經由微陣列方法來觀察脾臟與肝臟基因表現之改變。而於肝臟組織中，則可觀察到細胞膜上蛋白質與受體(receptor)之基因改變。近期吾等以大白鼠之出血性模式應用DNA微陣列發現休克與輸液復蘇，其基因體變化甚為相似，僅有6-磷酸萄萄糖水解脢 (glucose-6-phosphate dehydrogenase) 於出血性休克及輸液治療後24小時，其活性呈現有意義減少。因此，glucose-6-phosphate dehydrogenase可能是出血性休克對輸液治療反應的重要表現基因。6-磷酸萄萄糖水解脢為細胞中關鍵性調節脢，主要於細胞內氧化還原反應提供還原NADPH以維細胞恆定，於肝細胞中十分豐富。麩氨基硫(glutathione)也是肝臟中主要用於清除過氧化基，也是肝細胞抗過氧化基 (antioxidant)能力的指標。6-磷酸萄萄糖水解脢與麩氨基硫兩者，也於五碳糖磷化代謝途徑(pentose phosphate pathway)中關係十分密切，一起對抗過氧化基。因此6-磷酸萄萄糖水解脢也被吾等推論為出血性休克後與輸液復蘇中一個樞紐性代謝物，值得進一步探討。方法：本實驗之對象為雄性大白鼠，共分三組，第一組：控制組，第二組：休克組，第三組：休克及復甦組。每組又分為6小組(實驗開始0小時、1.5小時、4小時、8小時、12小時、24小時)，共108隻大白鼠參與實驗。出血性休克採用抽血2.5毫升血液╱公斤體重，並維持固定平均動脈壓為40+5毫米汞柱達一小時。復甦組給予10毫升血液╱公斤體重之乳酸林格液使穩定。實驗結束後給予安樂死，同時將大白鼠由心臟抽血並且將肝臟組織取下。取出之肝臟組織及血液立即放入-80℃液態氮急凍，再保存於-80℃之冰箱，直到接受進一步實驗之分析。血中細胞激IL-6，IL-10及6-磷酸萄萄糖水解脢活性，麩氨基硫(氧化態及還原態)予以檢測。肝組織麩氨基硫麩氨基硫(氧化態及還原態)也予以檢測。結果與討論：在我們的大白鼠創傷出血休克模式中，血液 G6PD活性在休克組及休克及復甦組中早期即增加並維持較控制組為高之活性。在休克組及控制組及休克+復甦組及控制組之間呈現顯著的差異；而復甦液給與，並未在24小時讓血液內G6PD活性增加的情形有所改變或消失。創傷出血休克改變大白鼠血液內氧化壓力狀態；在我們的大白鼠創傷出血休克模式中，血液中的還原型麩氨基硫(GSH)及血液中的GSH╱GSSG 比值在休克發生後不久即反應此狀態早早走低並持續了12-24小時。而復甦液給與，改善了血液中部分氧化的壓力。使得血液中的GSH╱GSSG 比值在實驗24小時內介於休克組及控制組之間。經由創傷出血休克亦改變了肝臟組織中的氧化壓力，但是肝臟組織對於此狀態其還原型麩氨基硫(GSH)及肝臟組織中的GSH╱GSSG 比值改變之反應殊於血液；肝臟組織的還原型麩氨基硫(GSH)先下降，直到實驗8-12小時點，看到肝臟組織中的還原型麩氨基硫(GSH)及肝臟組織中的 GSH╱GSSG 比值快速增加。相較於休克組，復甦液給與使得休克+復甦組並未出現此情形。發炎細胞激素IL6及抗發炎細胞激素IL10在創傷出血休克之後，最早的幾個小時內顯著增加，但是他們在實驗8-12小時點之後消失了。而復甦液給與使得休克+復甦組並未出現發炎細胞激素IL6增加之情形；然而在復甦液給與之後，抗發炎細胞激素IL10仍有增加，但幅度較休克組不明顯。結論：創傷出血休克改變血液中及肝臟組織中氧化的壓力情形。
Trauma-induced massive hemorrhage lead to development of tissue hypoxemia and ischemia╱reperfusion injury. Cellular injury and oxidative stress (oxygen free radical generation (ROS)) initiate post-injury priming of neutrophils and subsequent severe SIRS response. During the hemorrhage shock with or without immediate resuscitation, rat animal model, we had observed that the up-regulation of G6PD gene expression disappeared after immediate resuscitation compared with no immediate resuscitation group. It was also reported that incidence of sepsis increased and monocyte functions was altered in severely injured type-A-G6PD deficienct African American trauma patients. We hypothesize that G6PD is the important enzyme in the catalysis reaction of pentose phosphate pathway in which provides reducing power in the form of NADPH for reduction of GSSH to GSH and produces Ribose sugar as precursors in the biosynthesis of number of molecules, such as ATP, CoA, NAD, FAD,RNA and DNA. So G6PD may play an important role for energy providing in the early shock mechanism and subsequent immune response, development of MOF, especially in early stage. Methods: 108 mature SD, male, rats will be randomized into three groups (Gr.I: control group; Gr.II: hemorrhagic shock without resuscitation(HS); Gr.III: hemorrhagic shock with resuscitation(HS+R)). Rat hemorrhagic shock model ╱c fixed pressure model would be performed . The animals will be sacrificed by euthanasia and the whole blood and liver tissue will be collected at 0,1.5, 4, 8, 12, and 24 hours (each subgroup, 5-7 rats per time point) after the initiation of experiment. The whole blood sample and liver tissue will be collected and frozen until further studies. Blood G6PD activity test, liver tissue GSH and GSSG, whole blood GSH and GSSG, plasma inflammation cytokine IL6 and anti-inflammation cytokine IL10 would be tested. Result and Discussion: In our rat traumatic hemorrhagic shock model, G6PD activity increased early in HS group and HS+R group rats, compared with control group rats. There is significant difference of blood G6PD activity between shock group and control group and between HS+R group and control group. Resuscitation intervention dose not alter the increasing response of blood G6PD activity. Shock episode induced change of blood oxidative stress status and whole blood GSH and whole blood GSH╱GSSG ratio responded and went down early; it lasted for12-24 hours. Resuscitation intervention improved partial oxidative stress in blood (it made curve of whole blood GSH╱GSSG ratio between control group and shock group.) Oxidative stress condition also changed by shock episode in liver tissue and its response to the oxidative stress was differ from the whole blood. Increase of liver tissue GSH noted but delayed till the time-8-12-hour point. 5 Resuscitation intervention did not alter or change the liver tissue GSH in the early 12 hours, compared with the control group.Inflammation cytokine IL6 and anti-inflammation cytokineIL10 increased markedly after shock episode in the early several hours but they disappeared after the time-12-hour point. Resuscitation intervention made the increase of IL6 disappeared after shock episode in the early several hours. |
