敗血症時大鼠心室各分區中蛋白激酶C同功酶角色之探討

The Roles of Protein Kinase C Isoforms in Various Regions of Rat Ventricles during Sepsis

梁馨仁(Hsin-Jen Liang)

指導教授 ： 楊秀蘭

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

敗血症?vsepsis?w是一種由於身體受到感染而引起全身性反應的臨床症狀，主要是受到各種促發炎(pro-inflammatory)及抗發炎(anti-inflammatory)路徑的調節，當兩種路徑無法維持平衡時，則會伴隨著續發性的多重器官衰竭(multiple organ failure)。在敗血症時，位於心臟中的酵素與接受器系統會有所損傷，而使其收縮力減弱，最後導致心臟功能失調(cardiac dysfunction)或甚至死亡。於有關的研究報告中亦指出心肌細胞的凋亡(apoptosis)是造成心因性敗血性休克之主要病因。在粒腺體路徑的細胞凋亡過程中，學者發現：去磷酸化的助細胞凋亡蛋白Bad可由細胞質轉位至粒腺體上，因而導致細胞凋亡。然而，目前對於敗血症促使心臟功能失調的作用機制仍瞭解有限。
根據我們先前的研究結果顯示，在早期敗血症時，大白鼠心室中細胞質部分的蛋白激酶C活性增加，而細胞膜部分則無變化，而在晚期敗血症時，細胞質與細胞膜部分的蛋白激酶C活性皆無改變。此外，在不同的生理及病理情況下，心臟中各個區域有其獨特的功能及反應。截至目前為止，蛋白激酶C(PKC)至少有十二種同功酶已被確定，在改變心臟收縮力、肥大反應以及心肌缺血的耐受力等細胞訊息路徑，分別扮演不同的角色。亦有文獻證實，蛋白激酶C同功酶對於細胞的凋亡具有抑制性或刺激性之影響。因此，本研究目的主要是在探討敗血症時大鼠心室各分區中蛋白激酶C同功酶之角色。
本研究是採用大白鼠以盲腸結紮刺孔術(cecal ligation and puncture, 簡稱CLP)來引發敗血症之動物模式。實驗動物分成三組：控制組、早期敗血症組以及晚期敗血症組。早期敗血症組和晚期敗血症組分別是指在進行盲腸結紮刺孔術後之九小時與十八小時犧牲實驗動物，取出心臟並將其分為室中膈和左、右心室等三個區域。蛋白激酶C同功酶與助細胞凋亡蛋白Bad的蛋白質表現量先由西方點墨法分析，再以電泳膠片影像處理系統(densitometer)定量之。心肌細胞凋亡的組織型態則由TUNEL染色法確定。而粒腺體的超顯微結構是藉由電子顯微技術觀察。本研究實驗結果：(1) 在正常生理情況下的大鼠心室各分區中，蛋白激酶C同功酶α、β、δ、ε、ζ、ι、λ以及μ於細胞質與細胞膜等部分，呈現不一致的分佈表現。其中，特別是蛋白激酶C同功酶ε (PKC??)的分佈，在心室各分區中，細胞膜部分的表現皆顯著多於細胞質部分，故之後實驗則著重於探討此同功酶在敗血症時，於心室各分區中所扮演之角色。(2) 在敗血症的病理情況下，於大鼠室中膈及左心室等分區中，蛋白激酶C同功酶ε由細胞質轉位至細胞膜的活化隨著病程的進展而減少。(3) 在大鼠的室中膈及左心室等分區中，心臟細胞凋亡的程度與Bad由細胞質至粒腺體的轉位亦隨著敗血症的發展有所增加。(4) 在大鼠的室中膈及左心室等分區中，隨著敗血症的病程發展，越來越多粒腺體出現腫脹(swelling)的病理性變化，而粒腺體嵴(crista)的排列亦隨之呈現病態的紊亂(disarray)或甚至是無法分辨。
綜合上述之研究結果，我們發現在室中膈與左心室等分區，於敗血症的病理情況下，可能是因為蛋白激酶C同功酶ε的轉位活化減少，以致助細胞凋亡蛋白Bad之磷酸化亦減少，而使去磷酸化的Bad由細胞質轉位至粒腺體，造成粒腺體的結構異常，最後導致心肌細胞的凋亡。因此，我們建議蛋白激酶C同功酶ε的區域特異性之不活化，可能是造成敗血症時心臟損傷的重要原因。

關鍵字

敗血症 ； 助細胞凋亡蛋白Bad ； 心臟功能失調 ； 細胞凋亡 ； 蛋白激?C同功?ε

並列摘要

Sepsis is a clinical syndrome that results from the systemic response of the body to infection and is characterized and modulated by various pro-inflammatory and anti-inflammatory pathways. If homeostasis cannot be maintained, progressive and sequential dysfunction of various organ systems can occur. In the heart, enzyme/receptor systems have been reported to be damaged during sepsis, causing a decrease of contractility and leading to cardiac dysfunction and even death. Cardiomyocyte apoptosis contributes to the cardiogenic pathology of the septic shock. In the mitochondrial pathway, dephosphorylated Bad was found in the mitochondria and that has been implicated in apoptosis. However, the underlying molecular mechanisms for cardiac dysfunction have not yet been fully understood during sepsis.
Our previous results showed that cytosolic protein kinase C (cPKC) was activated in rat ventricles during the early hyperdynamic phase of sepsis, whereas membrane-associated protein kinase C (mPKC) activity was unchanged. During late sepsis, both cPKC and mPKC activities remained unchanged. Moreover, differential functions and responses are showed in various regions of heart under the state of physiology and pathology. For the moment, at least 12 protein kinase C (PKC) isoforms have been identified and may play different roles in cell signaling pathways leading to changes in cardiac contractility, the hypertrophic response, and tolerance to myocardial ischemia. Furthermore, the PKC isoforms have been shown to exert both inhibitory and stimulatory influences on apoptosis. Therefore, the present study was continuously investigated the roles of PKC isoforms in various regions of rat ventricles during sepsis.
Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 hr after CLP, respectively. Ventricular septum, Left ventricle, and right ventricle were extracted, respectively. The protein contents of various PKC isoforms and Bad were quantified by Western blot and densitometric analysis. Myocardial apoptosis pattern was detected by terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling (TUNEL). Ultrastructure of mitochondria was observed by electron microscopy. Our results showed: (1) PKC??, ??, ??, ??, ζ, ι, λ, and μ expressions were unevenly distributed in normal rat ventricles. Among them, the expression of membrane-associated PKC?? (mPKC??) was more than cytosolic PKC?? (cPKC??) in the septum, right and left ventricle under physiological condition; (2) While the PKC?? translocated from cytosol to membrane was decreased in left ventricle and septum during the progression of sepsis; (3) Myocardial apoptosis and Bad translocated from cytosol to mitochondria were increased in left ventricle and septum during the development of sepsis. (4) Mitochondria became swelling and its crista was disarrayed in left ventricle and septum during sepsis.
In conclusion, the PKCe translocation is decreased in septum and left ventricle that may decrease phosphorylation of the pro-apoptotic protein Bad. Then, the Bad translocated from cytosol to mitochondria and causing ultrastructure deformity of mitochondria. Finally, myocardial apoptosis is increased during the progression of sepsis. Therefore, we suggest that the area-specific inactivation of PKCe may contribute to the cardiac damage during sepsis.

並列關鍵字

sepsis ； cardiac dysfunction ； apoptosis ； PKCε ； Bad

參考文獻

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