Figure 4.

Effects of EGb761 on neuronal HO-1 protein expression in mouse primary neuronal cultures. A, Cultured neurons were pretreated for 1 hour with the protein synthesis inhibitor cycloheximide (CHX) or the mRNA inhibitor actinomycin D (ATD) in the concentrations shown before having 100 μg/mL EGb761 added to the culture medium for an additional 3 or 6 hours. The top panel shows the effect of the various drug regimens on HO-1 protein expression. The bottom panel shows actin expression in the same blot to indicate similar protein loading in all lanes. B, Primary neurons cultured for 14 days were pretreated with 10 μmol/L CHX or vehicle for 1 hour before being exposed to 100 μg/mL EGb761 or vehicle for 6 hours. Then cells were rinsed and incubated with fresh medium containing glutamate (30 μmol/L) or vehicle for an additional 18 hours. Each experiment was conducted in quadruplicate and repeated 3 times with different primary culture batches. Cell survival was estimated by the MTT assay and expressed as a percent of control viability. **P<0.01 compared with control groups.