The mechanisms of cannabidiol-induced apoptosis in primary immune cells
Date Issued
2012
Date
2012
Author(s)
Wu, Hsin-Ying
Abstract
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.
Subjects
apoptosis
cannabidiol
lipid raft
lymphocyte
microglia
oxidative stress
thiol depletion
Type
thesis
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