脂多醣 及 歐苷菊 引發內皮細胞鈣離子傳輸功能擾動之探討

Perturbations of membrane Ca2+ transport in endothelial cells treated with Lipopolysaccharide and Parthenolide

蔡天堯(Tien-Yao Tsai)

指導教授 ： 婁世亮

中原大學/工學院/生物醫學工程研究所/博士(2016年)

https://doi.org/10.6840/cycu201600472

摘要

內皮細胞不僅屏障血管組織與血液，還具有調節血管張力與維持血壓生理恆定等功能。內皮細胞分泌血管擴張因子（EDRF），其中最知名及重要的是一氧化氮（NO）；血管擴張活化物刺激內皮細胞細胞膜上的離子通道，引發鈣離子進入細胞內，並通過 三磷酸肌醇（IP3）等信使，將鈣離子由細胞內儲存器釋放到細胞內液，而細胞內鈣離子儲存器的清空，觸發細胞膜上存儲調控性鈣離子通道（SOCC）開啟，進而維持內皮細胞內鈣離子的上升濃度；內皮細胞內鈣離子上升，是觸發一氧化氮分泌的主要因子，一氧化氮分泌，使血管平滑肌舒張而擴張血管。內皮細胞也分泌血管收縮因子（EDCF），可以引發血管收縮。這些因子對血管維持健康非常重要，其分泌一旦失去平衡，則會導致血管疾病的發生。敗血症常導致重症疾病與死亡，而脂多醣（Lipopolysaccharide, LPS），或稱內毒素(Endotoxin)，是導致敗血症的重要成因。脂多醣內毒素可以傷害內皮細胞功能，導致血壓生理失去平衡而導致敗血症的危害。我的研究報告發現，經由脂多醣內毒素處理過存活的老鼠大腦皮質內皮細胞 (bEND.3 cell)，給予活化物，引發細胞內鈣庫釋放反應仍維持正常；但當 脂多醣內毒素處理細胞時間超過3小時或更久，則會導致細胞膜上 存儲調控性通道鈣離子內流 (SOCE) 下降。經由實時定量PCR分析，發現 脂多醣內毒素處理15小時後之內皮細胞，其 存儲調控性鈣離子通道上主要的兩個蛋白分子STIM1及Orai1，其基因表現並沒有減少，而且脂多醣內毒素引發之細胞存儲調控性通道鈣離子內流之下降，可以被 水楊酸鈉(sodium salicylate) 及 SB203580很明顯的減緩。水楊酸鈉是 核因子κB (nuclear factor-κB; NF-κB) 的抑制物，而 SB203580 是 p38絲裂原活化蛋白激酶( p38 mitogen-activated protein kinases ; p38 MAPK) 的抑制物，這表明了細胞存儲調控性通道鈣離子內流的抑制，是經由p38 MAPK–NF-κB 路徑。藉由此發現，讓我們更了解敗血症引發的機轉，有助於未來開發相關藥物與更有效的治療途徑。經由上述的研究，我嘗試使用另一個藥物歐苷菊來抑制 NF-κB 路徑，但是我卻觀察到引發鈣離子訊號大幅度更複雜的擾動，故我接著設計進一步實驗來研究此藥物的影響機制。歐苷菊（Parthenolide），又名小白菊内酯，是一種由天然植物萃取的物質，已知有抑制癌細胞的效果，且許多相關臨床試驗已在進行中。此外，在動物實驗中，歐苷菊藉由抑制細胞核因子NF-κB（nuclear factor-κB），可以減少心肌缺血再灌流後梗塞的面積大小、發炎細胞的浸潤與氧化傷害，對缺血再灌流損傷證實有保護作用，然而中高濃度的歐苷菊會釋放氧化活性物質（reactive oxygen species,ROS），卻會讓粒線體膜電位崩潰而對細胞產生毒性。至於對一般非腫瘤細胞，歐苷菊對離子信號傳導的影響所知有限。我的研究，測試經由歐苷菊處理15小時，存活的老鼠大腦皮質內皮細胞 (bEND.3 cell)，發現ATP活化鈣離子內流 (ATP-triggered Ca2+ signal) 信號被放大，且出現非常慢的衰減。此現象證實細胞內鈣離子排空受到抑制。證據顯示歐苷菊引發的鈣離子排空抑制，是經由抑制細胞膜上的鈣離子幫浦(plasmalemmal Ca2+ pump)，但西方點墨法分析顯示鈣離子幫浦上的蛋白表達總量並沒有減少；而此鈣離子排空的抑制現象，可被加入salubrinal而減輕，salubrinal 已知是一種內質網壓力保護劑 (ER stress protector) ，故此抑制現象，可能是經由內質網壓力(ER stress)路徑導致的結果。由於歐苷菊是進行中臨床研究的抗癌症藥物，且具有開發為心肌保護藥物的潛在潛力，故詳細了解歐苷菊對內皮細胞鈣離子恆定(Ca2+ homeostasis)及鄰近血管平滑肌細胞張力調控的影響是很重要的。藉由此研究，讓我們對歐苷菊的複雜機轉有更多了解，有助於未來進一步成功利用歐苷菊相關化合物開發治療性藥物。

關鍵字

內皮細胞 ； 內毒素 ； 歐苷菊 ； 存儲調控性鈣離子通道 ； 鈣離子幫浦 ； 核因子κB ； p38絲裂原活化蛋白激酶 ； 內質網壓力

並列摘要

Endothelial cells (EC) do not only serve the barrier function between the blood and the vascular smooth cells, but also regulate hemostasis, vascular tone and vessel morphology/proliferation. EC release an array of vasorelaxants --- EC-derived relaxing factors (EDRF); the most prominent known relaxing factors include nitric oxide (NO). Release of nitric oxide (NO) is triggered by a rise in endothelial cell (EC) cytosolic Ca2+ concentration ([Ca2+ ]i) and is of prime importance in vascular tone regulation as NO relaxes vascular smooth muscle. Agonists could stimulate EC [Ca2+ ]i elevation by triggering Ca2+ influx via plasma membrane ion channels, one of which is the store-operated Ca2+ channel; the latter opens as a result of agonist-triggered internal Ca2+ release. EC also release vasocontracting factors called endothelium-derived contracting factors (EDCF). The latter include endothelin-1, isoprostanes and reactive oxygen species. A loss of balance between the secretion of EDRF and EDCF may lead to vascular diseases. Endotoxin (lipopolysaccharide, LPS) could cause sepsis, which is often the fatal cause in critically ill patients. One of the LPS-induced damages is EC dysfunction, eventually leading to perturbations in hemodynamics. I obtained data showing that LPS-challenged mouse cerebral cortex endothelial bEND.3 cells did not suffer from apoptotic death, and in fact had intact agonist-triggered intracellular Ca2+ release; however, they had reduced store-operated Ca2+ entry (SOCE) after LPS treatment for 3 h or more. Using real-time PCR, I did not find a decrease in gene expression of stromal interaction molecule 1 (STIM1) and Orai1 (two SOCE protein components) in bEND.3 cells treated with LPS for 15 h. LPS inhibitory effects could be largely prevented by sodium salicylate (an inhibitor of nuclear factor-κB; NF-κB) or SB203580 (an inhibitor of p38 mitogen-activated protein kinases; p38 MAPK), suggesting that the p38 MAPK–NF-κB pathway is involved in SOCE inhibition. During the investigation of the above project, I used parthenonide to inhibit NF-κB; however, substantial perturbations in Ca2+ signaling was observed. Therefore, another project was developed to investigate the actions of this drug. Parthenolide is a sesquiterpene lactone compound isolated from the leaves and flowerheads of the plant feverfew (Tanacetum parthenium). The anticancer effects of parthenolide have been well studied and this lactone compound is currently under clinical trials. Parthenolide is also a protective agent in cardiac reperfusion injury via its inhibition of nuclear factor-κB (NF-κB). Not much is known if this compound affects signal transduction in non-tumor cells. I investigated whether parthenolide affected Ca2+ signaling in endothelial cells, key components in regulating the vascular tone. In this work using mouse cortical microvascular bEND.3 endothelial cells, I found that a 15-h treatment with parthenolide resulted in amplified ATP-triggered Ca2+ signal; the latter had a very slow decay rate suggesting suppression of Ca2+ clearance. Evidence suggests parthenolide suppressed Ca2+ clearance by inhibiting the plasmalemmal Ca2+ pump; such suppression did not result from decreased expression of the plasmalemmal Ca2+ pump protein. Rather, such suppression was possibly a consequence of endoplasmic reticulum (ER) stress, since salubrinal (an ER stress protector) was able to alleviate parthenolide-induced Ca2+ clearance suppression. Given the current deployment of parthenolide as an anti-cancer drug in clinical trials and the potential usage of this lactone as a cardioprotectant, it is important to examine in details the perturbing effects of parthenolide on Ca2+ homeostasis in endothelial cells and neighboring vascular smooth muscle cells, activities of which exert profound effects on hemodynamics.

並列關鍵字

Endothelial cells ； Endotoxin ； Store-operated Ca2+ entry ； P38 MAPK ； NF-kB ； Parthenolide ； Ca2+ clearance ； Ca2+ pump ； Endoplasmic reticulum stress

參考文獻

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