Ceramide 誘發細胞死亡之分子訊息探討: GSK-3β 角 色之研究

Molecular Signaling of Ceramide-Induced Cell Death: Role of GSK-3β

陳偉立(Wei-Li Chen)

指導教授 ： 施純明

臺北醫學大學/醫學院/醫學科學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00157

摘要

研究指出引發創傷性腦損傷（traumatic brain injury, TBI），伴隨微神經膠及星狀細胞活化而引發神經發炎反應，釋放活性氧分子及發炎性介質，繼而引起ceramide的合成，ceramide (神經醯胺)為細胞膜的成份之ㄧ，且為一個重要的次級訊息傳遞因子(second messenger)，參與眾多訊息傳遞路徑之調控，並可能導致細胞凋亡(apoptosis)，亦發現ceramide的含量上升為造成細胞死亡的主要原因之ㄧ，ceramide可因實驗模式與條件之差異而造成不同的死亡型式，包括細胞凋亡(apoptosis)及細胞自噬 (autophagy)。當創傷性腦損傷發生時，除了造成神經細胞的損傷，同時也會造成腦血管內皮細胞傷害。Autophagy為細胞中蛋白質分解之機制，其在細胞死亡中所扮演的角色仍有許多爭議，研究發現，顯示autophagy可扮演保護性角色，使細胞於不佳環境時得以存活，若抑制autophagy則會導致
細胞凋亡。近期文獻指出TBI後細胞會進行autophagy，顯示autophagy可能在TBI所導致之細胞死亡中扮演相當重要的角色。Glycogen synthase kinase-3β (GSK-3β)為一種蛋白質激酶，且已被證實參與TBI疾病過程，但其與autophagy之相關性仍不明。因此，本論文以腦血管內皮細胞(cerebral endothelial cells, CECs)為細胞模式，利用ceramide來模擬TBI所造成次及傷害，探討ceramide所導致autophagy與細胞死亡之關連性，並研究GSK-3β在TBI的疾病過程中所扮演之角色。實驗結果顯示，以C6-ceramide處理腦血管內皮細胞後，利用annexin V/PI double stain及acridine orange進行流式細胞儀定量實驗，發現在30 μM及12 h 之前autophagy之比例隨劑量及時間而增加，之後則隨之降低；apoptosis的比例均持續上升。而利用immunoblotting及穿透式電子顯微鏡技術，可明顯觀察到LC3-II蛋白質量上升及autophagosome形成，證實C6-ceramide可誘使腦血管內皮細胞產生混合型細胞死亡模式。再以immunoblotting分別偵測時間點GSK-3β蛋白質磷酸化程度，證明在腦血管內皮細胞C6-ceramide促使GSK-3β活化。且腦血管內皮細胞經由預先處理鋰鹽(LiCl)、GSK-3β抑制劑(AR-A014418)及siRNA可有效抑制C6-ceramide所誘使的autophagy並促使apoptosis增加，而過度表現(overexpression)GSK-3β則明顯增加C6-ceramide 所導致的autophagy 並降低apoptosis ， 故證明GSK-3β 在C6-ceramide誘導細胞死亡過程扮演保護性角色。藉由本論文之究，有助釐清TBI所導致細胞死亡的訊息傳遞路徑，以期望在臨床上可提供一個新的治療方針。

關鍵字

創傷性腦損傷,細胞自噬,細胞凋亡,腦血管內皮細胞

並列摘要

Ceramide, a sphingomyelin-derived second messenger,mediates cellular signalsof cytokines such as TNFα that are rapidly produced in the brain in response tovigorous neuronal activity and tissue injury, which is generated in cerebral endothelialcells. This activation may contribute to the progression of tissue damage and matrix destruction leading to BBB failure after a traumatic brain injury. The role of
autophagy in cell death is controversial. It was reported that autophagy serves as a protective mechanism helping cells survive from nutrient deprivation, and cells undergo apoptotic cell death when autophagy is inhibited. It was published that autophagy was observed after TBI, indicating that autophagy may be involved in TBI-induced cell death. Glycogen synthesis kinase- 3β (GSK-3β) is a serine/threonine kinase and activation of GSK-3β was able to regulate autophagy.
However, the role of GSK-3β in TBI-induced autophagy seems to be obscure. Inthis study, using cerebral endothelial cells as experimental model, we plan to investigate the underlying mechanisms of ceramide-induced autophagy and apoptosis, and the role of GSK-3 β i n TBI. After treatment with C6-ceramide, the CECs underwent autophagy and apoptosis in a dose- and time-dependent manner, as
revealed by acridine orange and Annexin V/PI double staining methods on a flow cytometer, respectively. Of which, the autophagy was confirmed by the formation of
autophagosomes and processing of LC-3, hallmarks of autophagy, using electron microscopy and immunoblotting, respectively. To demonstrate the involvement of glycogen synthase kinase-3β(GSK-3β), we employed LiCl, a selective inhibitor of GSK-3β, AR-A014418, and the siRNA technique to knockdown the expression of GSK-3 β , both of which resulting in decrease of the percentage of
C6-ceramide-induced autophagy and increase apoptosis. Following this line, cells harboring the GSK-3βgene by transient transfection were more sensitive to exposure
C6-ceramide, suggesting that GSK-3 β plays a crucial role in regulating the C6-ceramide-induced autophagy and apoptosis. Executing of this study will expand our knowledge regarding the signaling mechanism of TBI-induced cell death and may serve a new strategy for clinical therapy of TBI.

並列關鍵字

Traumatic brain injury (TBI)、C6-ceramide、autophagy、apoptosis、GSK-3β、cerebral endothelial cells(CECs)

參考文獻

Abbott N. J. Astrocyte–endothelial interactions and blood–brain barrier permeability. J. Anat. 200: 629–638, 2002.

Abbott N. J., Ronnback L. and Hansson E. Astrocyte–endothelial interactions at the blood–brain barrier. Nat. Rev., Neurosci. 7: 41–53, 2006.

Adelson P. D., Whalen M. J., Kochanek P. M., Robichaud P. and Carlos T. M. Blood brain barrier permeability and acute inflammation in two models of traumatic brain injury in the immature rat: a preliminary report. Acta Neurochir. Suppl. 71: 104-106, 1998.

Baethmann A., Eriskat J., Stoffel M., Chapuis D., Wirth A. and Plesnila N. Special aspects of severe head injury: recent developments. Curr Opin Anaesthesiol 11: 193–200, 1998.

Baldwin S. A., Fugaccia I., Brown D. R., Brown L. V. and Scheff S. W. Blood-brain barrier breach following cortical contusion in the rat. J. Neurosurg. 85: 476-481, 1996.