Figure 7. Radical scavenging activity and anti-oxidative effect of NecB. (A) Radical scavenging effects of various concentrations of NecB and two antioxidant controls, Tempo and NAC, were examined by incubation with DPPH reaction mixture, in which DPPH radical was induced by sonication. Percent scavenging activities of NecB were calculated and are plotted as the means ± standard deviations of at least three experiments. *P<0.05, **P<0.01, and ***P<0.001 compared with vehicle-treated control. (B) Young and old HDFs were pretreated with vehicle (PBS) or 100 μM H2O2 for 4 h and then treated with NecB, Tempo, or NAC for 24 h. The ROS level was measured by multiple plate reader after staining with DCF-DA. (C) Young and old HDFs were treated with NecB for 0, 24, or 48 h and the cellular levels of antioxidant enzymes (catalase, SOD I and SOD II) and β-actin were assessed by western blotting. The band density of the immunoblot was examined by densitometry and normalized to β-actin in each lane. The relative protein levels were calculated and plotted as the mean ± SEM. (D) Activation of the AMPK pathway reduced intracellular ROS levels A: AMPK was involved in the Nectandrin B-induced reduction in ROS levels. HDFs were transfected with AMPK siRNA and then treated with Nectandrin B in the absence or presence of palmitic acid for 24 h. The effectiveness of AMPKα knockdown was examined by anti-AMPKα antibody. ROS were detected by DPPH assay. Data represent the means ± SE (n = 3). **P < 0.01 ***P < 0.001. AMPKα siRNA prevented Nectandrin B-induced reduction of ROS.