大麻二酚(Cannabidiol)對抗體生成反應和T細胞活化與凋亡之影響

Abstract

大麻是一個被廣泛濫用的毒品，其主要成份為結構類似的大麻生物鹼（cannabinoids）。其中含量較豐富的有四氫大麻酚(Δ9-tetrahydrocannabinol；THC)，是大麻中樞活性的主要成份；另一成分大麻二酚(cannabidiol；CBD)則是不具中樞活性的主要成份。許多文獻均指出大麻具有明顯的免疫調節作用，影響包括in vivo 及in vitro的各種免疫反應。其中THC的作用已有大量的文獻報告。近年來CBD的研究逐漸受到重視，相關研究指出CBD具有許多藥理活性，包括抗痙攣、抗氧化、抗發炎、保護神經、改善關節炎的症狀以及抗化療的嘔吐等，顯示CBD是大麻生物鹼中一個頗具應用潛力的大麻天然成分。然而，CBD是否對免疫系統具有免疫毒性，文獻報告稀少。因此，本論文主旨在探討CBD對動物體抗體生成反應之免疫毒理學研究。 在本論文中，以卵蛋白(Ova)專一性抗體的生成反應，來觀察CBD對動物整體性的免疫調節作用。結果顯示，小鼠給予單一劑量的CBD對Ova專一性抗體生成反應呈現劑量相關性的抑制作用，在低劑量CBD（5 mg/kg）即有顯著的抑制效果。連續五天，每天一次個別給予腹腔注射CBD（5或20 mg/kg），結果也發現多重高劑量的CBD（20 mg/kg）具有顯著抑制Ova專一性抗體生成反應的作用。分析CBD抑制抗體生成反應的可能作用機制，結果顯示不論是in vivo或是in vitro投與CBD，皆對脾臟細胞分泌IL-2、IL-4和IFN-γ以及細胞的增生和代謝活性具一致性的抑制作用。但以ribonuclease protection assay (RPA)或RT-PCR分析細胞激素mRNA的表現，則沒有觀察到任何影響。顯示細胞激素的受抑制可能不是直接經由干擾細胞激素基因的表現。進一步探討CBD是否誘發脾臟細胞的凋亡反應，發現脾臟細胞以CBD處理6小時，代表凋亡後期的annexin V及propidium iodide陽性族群的細胞比率明顯上升，且CBD誘發脾臟細胞發生凋亡的作用呈現濃度相關性和時間相關性，接受CBD處理的細胞也出現凋亡反應典型的DNA laddering現象。進一步以RPA分析bcl family 調節細胞凋亡因子mRNA在脾臟細胞的表現情形，結果顯示CBD增加bak和bax等促進凋亡因子mRNA的表現。 綜合本論文的實驗結果，CBD的可能作用機制是經由促進bak和bax的表現而誘發脾臟細胞發生凋亡，導致脾臟細胞活性的下降，造成IL-2、IL-4和IFN-γ等細胞激素分泌的減少，進而導致抗體生成反應受到抑制。本論文的研究結果顯示不具中樞活性的CBD對動物體的抗原專一性抗體生成反應和T細胞活性具有顯著的免疫毒性作用。

Marijuana is one of the commonly abused drugs worldwide. The primary active components of marijuana are cannabinoids, such as Δ9-tetrahydrocannabinol (THC) which is psychoactive. In contrast to THC, cannabidiol (CBD) is another abundant cannabinoid that is nonpsychroactive. A line of evidence indicates that THC exhibits profound immunomodulatory activities in a variety of in vivo and in vitro experimental systems. Recently, a number of pharmacological activities possessed by CBD have been reported, including anticonvulsive, anxiolytic, antipsychotic, neuroprotective, antinausea and antirheumatoid effects. Thus, the potential pharmacological application and toxicity of CBD deserve further investigation. In light of the widely demonstrated immunomodulatory properties of cannabinoid compounds, the objective of the present studies was to investigate the effects of CBD on the immune system. The results demonstrated that administration of ovalbumin (Ova)-immunized BALB/c mice with single dose of CBD (5-20 mg/kg) markedly suppressed the serum concentrations of Ova-specific immunoglobulins in a dose dependent manner. Five consecutive daily injections of mice with the high dose of CBD (20 mg/kg) also produced the same inhibitory effect, whereas low dose of CBD (5 mg/kg) did not. To elucidate the possible underlying mechanisms by which CBD suppressed antibody production, the effects of CBD on cytokine secretion, proliferation and metabolic activity of splenocytes were examined. Both in vivo and in vitro treatment of splenocytes with CBD significantly inhibited IL-2, IL-4 and IFN-γ secretion, concanavalin A-induced proliferation, and metabolic activity of splenocytes. However, the expression of cytokine mRNA was not influenced by CBD treatment, suggesting that CBD-induced inhibition of cytokine secretion is not mediated directly by the suppression of cytokine gene expression. As splenocyte proliferation and metabolic activity were suppressed by CBD, the present studies investigated whether CBD could induce apoptosis of splenocytes. The results of flow cytometric analysis demonstrated that CBD increased the population of splenocytes that were double positive with annexin V and propidium iodide, indicating the induction of late apoptotic responses of cells by CBD. Furthermore, typical DNA laddering was observed in splenocytes treated with CBD. Further mechanistic studies showed the increased mRNA expression of bak and bax in CBD-treated splenocytes. Collectively, results of the present studies demonstrated the immunotoxicity of CBD in vivo and in vitro. CBD suppressed the antibody production by Ova-immunized mice, that is mediated, at least in part, by the inhibition of T-cell functions.