大麻二酚引起人類單核球氧化壓力與細胞凋亡之機制

Abstract

大麻二酚為一萃取自Cannabis sativa不具有中樞活性的天然生物鹼，具有抗發炎及免疫調節等藥理作用。目前已知大麻二酚對於多種癌化細胞及初代淋巴球、胸腺細胞以及單核球等具有促進凋亡的作用，且和氧化壓力有關。本研究目的為進一步探討大麻二酚造成人類單核球氧化性壓力以及引起細胞凋亡的機制。與先前報告結果一致，大麻二酚對於新鮮分離出的人類單核球具有凋亡效果，且具有時間與濃度相關性。時間相關性的實驗結果顯示，在給予大麻二酚後1-2小時會造成細胞內活性氧化物的增加。進一步比較後發現，在給予細胞大麻二酚5分鐘即造成粒線體膜電位去極化，且在15分鐘後造成粒線體內膜中心磷脂（cardiolipin）的氧化。大麻二酚也會造成粒線體中的細胞色素c（cytochrome c）釋放至細胞質，更加證明粒線體為大麻二酚的首要標的。除此之外，共軛膠顯微影像顯示，細胞內的氧化物螢光探針2’,7’-dichlorofluorescein與粒線體的螢光探針MitoTracker位置重和。流式細胞儀分析顯示，粒線體中鈣黃綠素（calcein）的螢光強度明顯減弱，證實大麻二酚會引起粒線體滲透性轉變。大麻二酚引起的凋亡和粒線體膜電位下降受到粒線體滲透性孔（mitochondrial permeability transition pore, MPTP）抑制劑環孢靈（cyclosporine A）的回復，但不受到鈣調去磷酸酶（calcineurin）抑制劑FK506的影響。另外，環孢靈也可抑制大麻二酚引起的粒線體心磷脂氧化和粒線體滲透性的轉變。綜合上述，大麻二酚引起人類周邊血液單核球的氧化性壓力和凋亡作用為粒線體所媒介，且為粒線體滲透性孔依賴性機制。

Cannabidiol (CBD), the major non-psychotropic cannabinoid contained in the plant Cannabis sativa, possesses a number of promising pharmacological activities, such as anti-inflammation and immunomodulation. CBD is known to induce apoptosis in both transformed and primary immune cells, including lymphocytes, thymocytes and monocytes, through oxidative stress-related mechanisms. The objective of this study was to investigate the underlying mechanisms for CBD-induced oxidative stress and apoptosis in monocytes. Consistent with previous results, exposure of freshly isolated human monocytes to CBD induced apoptosis in a time- and concentration-dependent manner. Time-course analyses revealed the induction of intracellular reactive oxygen species (ROS) at 1-2 h post CBD exposure. By comparison, CBD rapidly elicited the depolarization of mitochondrial membrane potential within 5 min, and the oxidation of cardiolipin, a major lipid component of the mitochondrial inner membrane, within 15 min of exposure. CBD also induced the release of cytochrome c from mitochondria to the cytosol, confirming that mitochondria are targeted by CBD. Furthermore, results from confocal microscopy demonstrated that the intracellular ROS measured by 2’,7’-dichlorofluorescein co-localized with the mitochondrial probe MitoTracker in CBD-treated monocytes. Flow cytometric analyses revealed that CBD induced the mitochondrial permeability transition (MPT) as evidenced by a marked decrease in the mitochondrial fluorescence of calcein. CBD-mediated apoptosis and mitochondrial depolarization were significantly attenuated in the presence of the mitochondrial permeability transition pore (MPTP pore) inhibitor cyclosporin A, but not affected by the calcineurin inhibitor FK506. Cyclosporin A also prevented cardiolipin oxidation and MPT induced by CBD. Taken together, the present study suggests that CBD may act at the level of mitochondria to initiate oxidative stress and apoptosis via MPTP-dependent mechanisms in human peripheral monocytes.