Figure 4.

The effects of cannabidiol (CBD) on the H₂O₂-evoked changes in reactive oxygen species level (ROS, (A)), mitochondrial membrane potential (MMP, (B)), caspase-3 activity (C), and cytotoxicity (D) in primary neuronal cell cultures. (A) ROS production was assayed with a CM-H₂DFFDA probe, as described in details found in the Material and Methods section. (B) MMP measurement was performed after 6 h of treatment of cells with CBD (0.01–5 μM), NAC (1 mM), and H₂O₂ (0.2 mM) employing a TMRE fluorescence probe. (C,D) Caspase-3 activity and cytotoxicity measurements were performed in cells treated for 9 h with CBD (0.01–5 μM) and H₂O₂ (0.2 mM). A caspase-3 inhibitor, Ac-DEVD-CHO (Ac, 20 μM), was used as a positive control to the assay. After treatment, in cell lysates, caspase-3 activity (C) was measured using the fluorogenic substrate Ac-DEVD-AMC, and cytotoxicity was assessed in the cell culture medium (LDH test, (D)). Data after normalization to vehicle-treated cells (100%) are presented as a mean ± SEM. * p < 0.05 and *** p < 0.001 vs. vehicle-treated cells; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. H₂O₂- treated cells.