大麻二酚引起人類初代單核球細胞凋亡之機制

Abstract

大麻二酚為一不具有中樞活性的天然大麻生物鹼，目前已知對許多免疫細胞具有凋亡作用，包含初代和癌化的腫瘤細胞。文獻指出人類周邊血液單核球經體外培養72小時後，以大麻二酚處理並不會引起凋亡。因此，本研究的主旨為探討大麻二酚對於新鮮分離與培養過的單核球的凋亡作用，並比較其異同。結果發現大麻二酚顯著引起新鮮單核球凋亡，此作用具有濃度相關性；然而對經過72小時培養的單核球，則無凋亡作用。分析大麻二酚對於新鮮單核球造成凋亡的機制，發現大麻二酚引起細胞內活性氧化物的增加，包括超氧自由基，過氧化氫和氫氧自由基。抗氧化物質包含N-乙醯-L-半胱胺酸、過氧化氫酶、丙酮酸鈉、雙甲基硫尿和4,5-二氫基-1,3-苯二磺酸，可明顯降低大麻二酚引起的單核球凋亡比例。比較新鮮與培養過的單核球之間的差異，可發現培養過的單核球有較好的抗氧化能力，因其具有較豐富的穀胱甘肽、超氧化物歧化酶和第一型血色素氧化酵素。觀察新鮮單核球在給予大麻二酚之後細胞內抗氧化物質的變化，由結果得知大麻二酚顯著地消耗新鮮單核球內的穀胱甘肽和超氧化物歧化酶。利用第一型血色素氧化酵素抑制劑抑制培養過單核球的抗氧化能力，可使其恢復對大麻二酚凋亡作用的感受性。由這些結果可得知抗氧化能力能決定單核球是否能抵抗大麻二酚引起的氧化性傷害，有關大麻二酚如何增加細胞內活性氧化物的機制仍有待釐清。使用類香草素受體1的拮抗劑可使大麻二酚造成的凋亡比例下降，推測大麻二酚有可能藉由此途徑誘發單核球凋亡。綜合以上結果可得知大麻二酚增加新鮮單核球中的活性氧化物並造成細胞凋亡，而培養過的單核球具有較佳的抗氧化能力，可抵抗大麻二酚產生的氧化性傷害和凋亡作用。

Cannabidiol (CBD), the major non-psychoactive cannabinoid, has been documented to induce apoptosis in a variety of immune cells, including primary lymphocytes and transformed lymphocytic and monocytic cell lines. In contrast, human peripheral blood monocytes precultured for 72 h have been reported to be insensitive to CBD-mediated apoptosis. The objective of this study was to investigate the pro-apoptotic effect of CBD on monocytes that were either freshly isolated or precultured for 72 h. The results showed that CBD markedly enhanced apoptosis of freshly isolated monocytes in a concentration-dependent manner, whereas 72 h-precultured monocytes were insensitive. Exposure of monocytes to CBD elicited an early production of reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide and hydroxyl radicals. Pretreatment of monocytes with antioxidants, including N-acetyl-L-cysteine, catalase, sodium pyruvate, dimethylthiourea and 4,5-dihydroxy-1,3-benzenedisulfonic acid, significantly attenuated the apoptosis induced by CBD. In addition, precultured monocytes contained a greater level of glutathione (GSH), superoxide dismutase (SOD) and heme oxygenase-1 (HO-1), as compared to the freshly isolated cells. CBD diminished the level of cellular GSH and SOD in fresh monocytes. Zinc protoporphyrin, a specific HO-1 inhibitor, restored the sensitiveness of precultured monocytes to CBD-mediated apoptosis. The antioxidant ability of monocytes may influence the apoptotic effect of CBD. The mechanism of CBD induced oxidative stress is unclear. The vanilloid receptor type 1 (VR1) antagonist capsazepine attenuated CBD induced apoptosis, suggesting that VR1 may be a potential target involved in CBD-mediated monocytes apoptosis. In summary, the present study demonstrated a contrasting pro-apoptotic effect of CBD between precultured and freshly isolated monocytes, which was closely associated with the antioxidative capability of the cells.