Involvement of p-CREB and phase II detoxifying enzyme system in neuroprotection mediated by the flavonoid calycopterin isolated from Dracocephalum kotschyi

Purpose: There is an increasing amount of experimental evidence that oxidative stress has a central role in the neuropathology of neurodegenerative diseases. It has been suggested that the loss of cell function results from the increased oxidative damage to proteins and DNA. Herein, we investigated the effect of a natural neuroprotective flavonoid, calycopterin, on H₂O₂-induced disruption of phase II detoxifying enzyme system and cAMP response element binding protein (CREB) phosphorylation.

Methods: PC12 cells were treated with 25, 50 and 100 μM of calycopterin for 3h, followed by adding H₂O₂ (150 μM) for 24 h. The extent of apoptosis was assessed by comet assay. The level of phosphorylated CREB, nuclear factor erythroid 2-related factor 2 (Nrf2), glutamylcysteine synthetase (γ-GCS) and heme oxygenase 1 (HO-1) were measured by western blot method. The concentration of glutathione (GSH) was determined in whole cell lysate using dithionitrobenzoic acid method. Superoxide dismutase (SOD) activity was measured by colorimetric assay.

Result: Morphological analysis of protection induced by calycopterin, determined by comet assay, showed that calycopterin reduced DNA in tail. We found that H₂O₂ decreased mitochondrial membrane potential (MMP), while, calycopterin prevented this decrease in MMP in presence of H₂O₂. In H₂O₂-treated cells, calycopterin also suppressed cytochrome C release to cytosol that is necessary for maintaining mitochondrial homeostasis in survived cells. Moreover, calycopterin, in presence of H₂O₂ inhibited the decrease caused by oxidative stress in stress-sensing transcription factors, CREB and Nrf2, which play an important role in antioxidant capacity of the cell. There was also an increase in γ-GCS and HO-1 levels in calycopterin pretreated cells. In the presence of H₂O₂, calycopterin inhibited decrease in GSH level and SOD activity.

Conclusion: We provided documentation of neuroprotective effect of a natural flavone, calycopterin, against H₂O₂-induced oxidative stress in differentiated PC12 cells by modulating the level of CREB phosphorylation and Nrf2 pathway.