Figure 1

CBD induces cell death of BV-2 microglial cells. (a) CBD dose-dependently induces cell death in BV-2 cells after 2 h treatment under serum-free conditions. Cell death is quantified as the increase in % counts in the subG1 phase (100%=total counts in all cell cycle phases: SubG1, G1, S, and G2/M). Significance of SubG1 counts compared with vehicle (ethanol 0.1%). CBD 5 μM, *P<0.001, n=3; CBD 10 μM, ^#P<0.001; n=5, one-way ANOVA with Bonferroni post hoc). (b) The effect of several antagonists on CBD (10 μM)-induced cell death. CBD-induced cell death was significantly enhanced by pretreatment with 7.5 μM GW9662 (GW; ^$P<0.01, n=3, one-way ANOVA with LSD post hoc) and was significantly attenuated by 5 μM ZM241385 (ZM; *P<0.002, n=3, one-way ANOVA with LSD post hoc) and by 7 μM, Cys A (CYSA; ^#P<0.001, n=4, one-way ANOVA with LSD post hoc), CBD-induced cell death was not affected by 0.5 μM SR144528 (SR; n=6). All antagonists were dissolved in DMSO (0.1% final concentration). (c) CBD (10 μM)-induced cell death was significantly reduced by pretreatment with 10 μM ruthenium red (RR; ^#P<0.05, n=3, one-way ANOVA with LSD post hoc), 10 μM WAY100635 (WAY; ^#P<0.05, n=3, one-way ANOVA with LSD post hoc). CBD-induced cell death was not affected by 100 ng/ml PTX (n=3), 1 mM NAC (n=5), or 50 μM L-NAME (n=3). All antagonists were diluted in water (excluding WAY100635 which was diluted in saline). (d) Cholesterol enrichment (50 μM, in sesame oil final concentration 0.1%) significantly reduced CBD (10 μM)-induced cell death (^#P<0.001, n=3–5, one-way ANOVA with LSD post hoc). All compounds tested didn’t induce cell death in the absence of CBD