Fig. 4
In vitro and in vivo anti-oxidative activity of PPE on DNCB-induced ROS. (a) Cellular ROS production was induced by 40 μM DNCB in HaCaT cells. To determine the effect of PPE on DNCB-induced ROS production, HaCaT cells were co-treated with DNCB and PPE for different time periods (3, 6, 12, 24 and 48 h). Intracellular ROS were stained with the H2DCFDA probe, and the fluorescence was visualized under a microscope. DNCB generated significant amounts of ROS in cells, which gradually disappeared with PPE treatment. After 48 h of PPE treatment, fluorescence intensity diminished to the basal level (normal group). DEX did not display anti-oxidative activity after 24 h of treatment. (b) Inhibitory effect of PPE on ROS generation in vivo. Ears of mice were pre-treated with cream (vehicle) alone, PPE or DEX in cream for 1 h before induction of ROS by topical application of 5% DNCB. Normal and 5% DNCB-treated groups were treated with PBS only or PBS with DNCB. Ear tissues were obtained after mouse euthanasia and stained ex vivo with 5 mM DHE in DMSO for 30 min. Fluorescence generated by ROS was visualized under a microscope. DNCB induced significant ROS production, which was almost completely abrogated by PPE