嗎啡促進人類臍靜脈內皮細胞的凋亡

Abstract

morphine是臨床常用的鴉片類opioid止痛藥。它是綜合性μ、δ、κ鴉片類受器作用藥物，主要對μ鴉片類受器μ-opioid receptor具有高親合性，在腦部即經由μ-1鴉片類受器達到止痛效果。以往對morphine的了解，也多著重於其對神經系統μ、δ、κ鴉片類受器的作用。然而，隨著身體其他系統鴉片類受器的發現，morphine在神經系統外的效用，已受到許多研究重視。例如，morphine作用在免疫系統immune system的鴉片類受器，會造成免疫細胞凋亡；對後天免疫功能acquired immune function及先天免疫功能innate immune function，產生抑制效果。如吞噬細胞phagocytic cell功能、淋巴細胞lymphoid cell功能、細胞激素cytokine製造等，皆受到抑制。在藥癮患者等，可能會增加感染的危險外。在癌症末期的疼痛控制，因常需依賴長期而大量的morphine治療；因此，morphine免疫抑制及加速腫瘤生長的可能性，也引起廣泛討論。 除了在免疫系統，近來血管內皮細胞vascular endothelial cell的鴉片類受器也被發現。研究顯示血管內皮細胞上具有μ-3鴉片類受器μ-3 opioid receptor，並且與NO釋放與血管擴張有管。這引發了morphine使用，對血管內皮系統影響的研究興趣。血管內皮系統與許多生理病理狀態，如血管阻力vascular tone的調節、發炎反應inflammation的控制、血液與組織間物質的交換permiability，凝血作用coagulation的調控、動脈粥狀硬化atherosclerosis的發生、血管新生angiogenesis的參與等，都息息相關。任何影響血管內皮細胞功能及存活的狀況，也會對這些生理病理現象發生影響。那到底morphine對血管內皮細胞有什麼作用？目前僅有少數有關這方面的研究。 最近一篇報告發現，morphine會促進體外實驗血管內皮細胞的凋亡，並增加血管內皮細胞通透性。在lipopolysaccharide引起的血管內皮細胞凋亡數量增加及通透性上升，morphine也會加成其作用。其他研究卻顯示，morphine會增進血管內皮細胞的增生；於臨床治療濃度下，對體外和體內血管新生實驗亦皆具促進作用，並使小鼠的乳癌腫瘤模式生長加速。這些零星報告綜觀來說，對於morphine對血管內皮細胞的影響，其是否引發細胞凋亡或促進細胞增生，並沒有清楚一致的結論。 為了釐清morphine對血管內皮細胞的作用，及μ鴉片類受器所扮演的角色，我們設計以人類臍靜脈內皮細胞HUVECs（Human Umbilical Vein Endothelial Cell）細胞培養的體外實驗來模擬，觀察不同濃度morphine，對血管內皮細胞增生或凋亡的影響；並探討其可能細胞凋亡作用機轉，抗細胞凋亡分子及促細胞凋亡分子之表現。此外，並加入μ鴉片類受器阻斷劑μ-opioid receptor antagonist，「naloxone」，來遮斷血管內皮細胞上的μ鴉片類受器；觀察morphine對於人類臍靜脈內皮細胞的作用，是否會因naloxone加入而被拮抗還原，以鑑別morphine的作用路徑，是否是經由μ鴉片類受器來傳遞： 將包括臨床治療範圍（血中濃度2~3500 nM）的不同濃度morphine（1~106 nM），加入人類臍靜脈內皮細胞中培養。以MTT Assay，分析其對細胞增生proliferation、細胞活性viability的影響。在細胞凋亡apoptosis方面，我們用Hoechst染色鏡檢觀察凋亡細胞型態morphalogy變化來做定性實驗；並且用Annexin-V染色，以流式細胞儀FACS Analysis來定量morphine對細胞凋亡的影響。對於可能的細胞凋亡路徑，我們以Western Blot來偵測Bax, Bak, Bcl2等細胞凋亡內在路徑intrinsic pathway相關因子的蛋白質含量。文獻指出，當細胞趨向凋亡時，促細胞凋亡分子如Bax、Bak，及抗細胞凋亡因子Bcl-2會有相互消長的情形，使Bcl-2/Bax比值變小。最後，我們並以不同濃度μ鴉片類受器拮抗劑naloxone，預先處理人類臍靜脈內皮細胞，來阻斷morphine對μ鴉片類受器的作用；2小時後再加入104nM的morphine，並重複以上實驗，以確認其作用路徑是否經由μ鴉片類受器。 在實驗中我們觀察到：morphine會抑制人類臍靜脈內皮細胞增生及活性，並有隨濃度增加而抑制程度增加的趨勢。並且隨著morphine濃度增加，人類臍靜脈內皮細胞HUVECs凋亡比例亦增加。同時，細胞凋亡促進因子Bax和Bak，及細胞凋亡抑制因子Bcl2，都會因morphine濃度增加而變化；Bcl-2/Bax平衡發生改變，其比值顯示趨向細胞凋亡。以ANOVA test統計分析後，其p值小於0.05，顯示具有統計學上的差異。這些，在加入μ鴉片類受器拮抗劑naloxone預先處理後，morphine對人類臍靜脈內皮細胞的作用會被阻斷。並且，在高濃度naloxone，比起低濃度nalxone，其阻斷作用更快發生，也更為明顯。 綜合以上，我們提出了一個結論：相對高劑量以上的morphine，會減少人類臍靜脈內皮細胞HUVECs的生長，並且促進人類臍靜脈內皮細胞的凋亡。這些作用，會隨著morphine濃度增加，使影響程度增加（dose-dependent）。並且morphine對人類臍靜脈內皮細胞的影響程度，會隨作用時間增長而變強（time-dependent）。而morphine促進人類臍靜脈內皮細胞HUVECs的凋亡，可能是經由增加促凋亡因子Bak和Bax的含量，使原本決定細胞生存或死亡的Bcl-2/Bax的平衡發生改變，進一步刺激粒腺體釋放Cytochrom-C，結合Apaf-1及Caspase9，經由細胞凋亡內在路徑intrinsic pathway，最終活化Caspase3，促使細胞凋亡的現象開始發生。以上這些morphine對人類臍靜脈內皮細胞的作用，至少部分是經由刺激μ鴉片類受器而發生；阻斷μ鴉片類受器，能夠有效阻斷morphine的作用。 對於體外人類臍靜脈內皮細胞培養模式的實驗結果，有助於我們模擬瞭解morphine對於體內血管內皮系統可能的影響。在臨床上藥癮、癌症、或是慢性疼痛的病人，在長期大量使用嗎啡類止痛藥下，對於其血管阻力、發炎反應、血管通透性、凝血功能、動脈粥狀硬化、血管新生等等，會有什麼作用、對其病程的可能影響，是我們未來努力研究的方向。這些結果對於現行疼痛治療方針的改進，將有重大的幫助。

Morphine is widely used for pain control. It is an combined μ, δ-, and κ-opioid agonist with high affinity to μ-opioid receptors. However, besides of its analgesic effects, little was known about the effects beyond the nervous system. With the discovery of opioid receptors outsides the nervous system, the old drug became a focus of investigation again. The immuno-suppression property of morphine was discussed. Both specific and non-specific immune systems were suppressed by stimulation of opioid receptors on immune cells. Although little studies concluded that immuno-suppression effects of morphine would be harmful to patients, accelerating infection and promoting tumor growth were suggested. Moreover, opioid receptors like μ- and δ-receptors were also discovered on endothelial cells. Increase of NO production were reported linked toμ3-opioid receptors, leading to vesoldillation. However, still little was known about the effects of morphine on the vascular endothelial system. Different studies led to converse results. One showed that morphine stimulates proliferation of endothelial cells in vitro and promotes angiogenesis and tumor growth in vivo. Meanwhile, the other showed that morphine potentiated lipopolysaccharide-induced apoptosis and permeability of vascular endothelial cells. All of these interest us to elucidate the effects and the molecular mechanisms of morphine on endothelial cells, and the possible role of μ-opioid receptors in the transduction pathway. For this reason, we established the in-vitro model of human umbilical vein endothelial cells (HUVECs) to figure out the effects of morphine on the vascular endothelial system. Different concentrations of morphine (1~106nM), covering the range of clinical therapeutic plasma concentration (2~3500nM), were investigated. In addition, naloxone, a μ-opioid receptor antagonist, pretreatment (103nM/104nM or 10nM/102nM) were added to block the μ-opioid receptors preceding morphine treatment. If the effects of morphine on HUVECs attenuated, they should be via μ-opioid receptors. MTT proliferation assay was performed to evaluate the cell viability. Microscopy with Hoechst stain was used to distinguish the morphology of apoptotic cells. Flowcytometry (FACS) with Annexin-V stain was performed to assay the ratio of apoptotic cells. Meanwhile, the amount of pro-apoptotic and anti-apoptotic factors in the intrinsic apoptosis pathway, such as Bax, Bak, and Bcl2, were analyzed by Western blot. The balance of pro-apoptotic and anti-apoptotic factors would decide the direction of cells to survival or apoptosis. Our study showed that morphine inhibited proliferation and promoted apoptosis of HUVECs in a dose-dependant manner. The effects of morphine on HUVECs became prominent while the concentration of morphine was above 103nM. There were statistical significance (P < 0.05), using ANOVA test. Besides, we found that the amounts of Bak and Bax increased as the concentration of morphine elevated, while the amount of Bcl2 remained unchanged. The ratio of pro-apoptotic factors, Bax and Bak, and anti-apoptotic factors, Bcl2, would lead the cells to apoptosis. Naloxone pretreatment blocked the effects of morphine on HUVECs mentioned above. The high-dose naloxone pretreatment was more effective than the low-dose naloxone preatment. In the high-dose naloxone pretreatment group, naloxone could almost totally block the effects of morphine. In summery, our results suggest that morphine decreases viability and increases apoptosis of human umbilical vein endothelial cells (HUVECs), which is mediated byμ-opioid receptors. And apoptosis of HUVECs induced by morphine occurs, at least in part, through stimulating the expression of pro-apoptotic factors in the apoptosis intrinsic pathway. Our results can provide more information about the effects of morphine on vascular endothelial system, and lead to the initiation of investigations about inflammation, permeability, coagulation, atherosclerosis, and angiogenesis in future.