大麻二酚誘發初代免疫細胞凋亡之機制探討

吳欣穎

doi:10.6342/NTU.2012.03016

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大麻二酚誘發初代免疫細胞凋亡之機制探討

The mechanisms of cannabidiol-induced apoptosis in primary immune cells

吳欣穎 , Ph.D　　Advisor：詹東榮　　

英文 DOI： 10.6342/NTU.2012.03016

凋亡；大麻二酚； caspase ； caveolin-1 ； HU-331 ；脂筏；淋巴球；微膠細胞；氧化性壓力； thiol流失； apoptosis ； cannabidiol ； caspase ； caveolin-1 ； HU-331 ； lipid raft ； lymphocyte ； microglia ； oxidative stress ； thiol depletion

Abstract │ Reference (120) │ Altmetrics

Abstract 〈TOP〉

大麻二酚是大麻生物鹼中主要不具中樞活性的成份，具有許多的藥理作用，包括免疫調節及抗發炎。本實驗的主旨在探討大麻二酚對初代免疫細胞，包括脾臟細胞及微膠細胞之促凋亡作用及其潛在之作用機制。在小鼠脾臟細胞，大麻二酚會誘發細胞凋亡、產生活性氧化物、降低細胞內glutathione（GSH）之含量，並且促進caspase-8的活化。上述大麻二酚之作用，在給予thiol類抗氧化劑N-acetyl-L-cysteine（NAC）後，即被回復。給予caspase-8的抑制劑也可以有效地回復大麻二酚誘發之凋亡作用，但是不影響大麻二酚造成的活性氧化物之生成。上述實驗結果說明大麻二酚於初代淋巴球細胞誘發之凋亡作用和氧化性壓力造成之caspase-8的活化密切相關。
由於大麻二酚已被證實可被微粒體中的酵素轉變成hydroxyquinone類的代謝物HU-331，並和細胞內的GSH形成鍵結物。為了進一步釐清大麻二酚誘發脾臟細胞凋亡之機制，本研究接著測試HU-331是否會經由造成脾臟細胞內GSH的流失而誘發凋亡。結果顯示HU-331會誘發脾臟細胞之凋亡及細胞內thiol和GSH的逐漸流失；給予thiol類抗氧化劑NAC及N-(2-mercaptopropionyl) glycine可以回復前述HU-331致凋亡及GSH流失之作用，而非thiol類的抗氧化物catalase及pyruvate則無效。在脾臟細胞也觀察到類似的結果，即大麻二酚的凋亡作用能被thiol類抗氧化劑回復，而非thiol類的抗氧化物則無效。上述結果說明大麻二酚可能是經由其活性代謝物HU-331造成細胞內thiol之流失而導致脾臟細胞的凋亡。
在小鼠初代微膠細胞，大麻二酚會誘發細胞凋亡及活化caspase-8及caspase-9，機制研究顯示抗氧化劑NAC和GSH、GPR55致效劑abnormal-CBD及vanilloid受體、CB1和CB2受體拮抗劑均無法回復大麻二酚所誘發之凋亡作用。相反地，脂筏破壞劑methyl-β-cyclodextrin (MCD)則可有效地降低大麻二酚誘發之微膠細胞凋亡和caspase的活化。此外，大麻二酚會誘發lipid raft coalescence，增加GM1 ganglioside 和caveolin-1表現，這些作用也可被MCD所回復。上述結果說明大麻二酚會經由誘發lipid raft coalescence、增加GM1 ganglioside和caveolin-1的表現而導致初代微膠細胞的凋亡。
綜合上述，本研究顯示大麻二酚分別經由thiol流失及脂筏相關之機制，誘發初代脾臟細胞及微膠細胞之凋亡，本研究首度證實大麻二酚會誘發不同之初代免疫細胞凋亡，拓展了大麻二酚促凋亡作用之範疇，並且對大麻二酚誘發免疫細胞凋亡之機制提供了新的見解，誘發免疫細胞之凋亡可能是大麻二酚免疫調節及抗發炎作用之一個重要機制。

Parallel Abstract 〈TOP〉

Cannabidiol (CBD), the major non-psychoactive cannabinoid in marijuana, exhibits a variety of pharmacological activities, including immunomodulatory and anti-inflammatory effects. The objective of the current study is to investigate the pro-apoptotic effect of CBD on primary immune cells, including murine splenocytes and microglial cells. In splenocytes, CBD markedly enhanced the apoptosis, elicited the production of reactive oxygen species (ROS), diminished the level of intracellular glutathione (GSH), and stimulated the activation of caspase-8, all of which were remarkably attenuated by the thiol antioxidant N-acetyl-L-cysteine (NAC). Pretreatment of splenocytes with caspase-8 inhibitor significantly attenuated CBD-mediated apoptosis, but not ROS production. Collectively, these results demonstrated that CBD-induced apoptosis in primary lymphocytes is closely associated with oxidative stress-dependent activation of caspase-8.
It has been reported that CBD can be metabolized by microsomal enzymes to cannabidiol hydroxyquinone (HU-331) that forms adducts with glutathione. To further investigate the mechanism of CBD-induced apoptosis in splenocytes, the present study tested the hypothesis that HU-331 could cause apoptosis via the depletion of thiols in splenocytes. HU-331 treatment resulted in splenocyte apoptosis and a gradual diminishment in the cellular thiols and glutathione. The HU-331-induced apoptosis and thiol diminishment were abrogated in the presence of thiol antioxidants, including NAC and N-(2-mercaptopropionyl) glycine, whereas the non-thiol antioxidants catalase and pyruvate were ineffective. Similar results were also observed in CBD-treated splenocytes, in which thiol antioxidants but not non-thiol antioxidants markedly attenuated CBD-induced apoptosis. These results suggest that HU-331 might be an active metabolite of CBD potentially contributing to the induction of apoptosis in splenocytes, and that the apoptosis is primarily mediated by the loss of cellular thiols. 
In primary microglia, CBD resulted in the induction of apoptosis and marked activation of both caspase-8 and -9. Mechanistic studies revealed that antioxidants, including NAC and GSH, the GPR55 agonist abnormal-CBD and specific antagonists for vanilloid, CB1 and CB2 cannabinoid receptors did not counteract the apoptosis induced by CBD. In contrast, methyl-β-cyclodextrin (MCD), a lipid raft disruptor, potently attenuated CBD-induced microglial apoptosis and caspase activation. In addition, CBD induced lipid raft coalescence and augmented the expression of GM1 ganglioside and caveolin-1, all of which were attenuated by MCD. These results suggest that CBD induces a marked pro-apoptotic effect in primary microglia through lipid raft coalescence and elevated expression of GM1 ganglioside and caveolin-1.
In summary, the present study demonstrated the pro-apoptotic effect of CBD in primary splenocytes and microglial cells, which was predominantly mediated by the thiol depletion and lipid raft-related mechanism, respectively. The present findings extend our current understanding on the spectrum of CBD-mediated apoptosis and provide new insights to the underlying mechanisms in primary immune cells. The induction of immune cell apoptosis may be a critical mechanism contributing to the immunomodulatory and anti-inflammatory properties of CBD.

Reference ( 120 ) 〈TOP〉

Andradas C, Caffarel MM, Perez-Gomez E, Salazar M, Lorente M, Velasco G, Guzman M and Sanchez C. The orphan G protein-coupled receptor GPR55 promotes cancer cell proliferation via ERK. Oncogene 30: 245-252, 2010.
連結：
Baczynsky WO and Zimmerman AM. Effects of delta 9-tetrahydrocannabinol, cannabinol and cannabidiol on the immune system in mice. II. In vitro investigation using cultured mouse splenocytes. Pharmacology 26: 12-19, 1983.
連結：
Bari M, Battista N, Fezza F, Finazzi-Agro A and Maccarrone M. Lipid rafts control signaling of type-1 cannabinoid receptors in neuronal cells. Implications for anandamide-induced apoptosis. J Biol Chem 280: 12212-12220, 2005.
連結：
Ben-Shabat S, Hanus LO, Katzavian G and Gallily R. New cannabidiol derivatives: synthesis, binding to cannabinoid receptor, and evaluation of their antiinflammatory activity. J Med Chem 49: 1113-1117, 2006.
連結：
Bidinger B, Torres R, Rossetti RG, Brown L, Beltre R, Burstein S, Lian JB, Stein GS and Zurier RB. Ajulemic acid, a nonpsychoactive cannabinoid acid, induces apoptosis in human T lymphocytes. Clin Immunol 108: 95-102, 2003.
連結：

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