3.5. Effects of MQL on ROS generation in CD4+ T cells
Phytochemicals have been well-known as antioxidants that can directly scavenge ROS, and are used to protect against inflammation36,37. Whereas phytochemicals can generate appropriate intracellular ROS, resulting in up-regulation of HO-1 expression38. Furthermore, intracellular ROS have been linked to ERK1/2 as cAMT-mediated ROS production can induce ERK1/2 phosphorylation via Ras activation39. Thus, we investigated intracellular ROS generated by MQL in CD4+ T cells using DCFH-DA or MitoSOXTM Red, and found that MQL increased intracellular and mitochondrial ROS levels (Fig. 5A and B) . Next, to investigate effects of intracellular ROS generated by MQL on the C-Raf-ERK1/2 signaling pathway, CD4+ T cells were cultured with MQL in the presence and absence of the antioxidant N-acetylcysteine (NAC). We found that MQL-induced C-Raf and ERK1/2 phosphorylation, as well as HO-1 expression, were suppressed by NAC treatment (Fig. 5C and D) . Taken together, these results suggest that MQL-induced ROS mediate Ras-Raf-ERK activation and HO-1 expression in CD4+ T cells.