Induction of Antioxidant Protein HO-1 Through Nrf2-ARE Signaling Due to Pteryxin in Peucedanum Japonicum Thunb in RAW264.7 Macrophage Cells

Abstract

This study focused on exploring the nuclear factor-erythroid-2-related factor (Nrf2) active compound to avoid oxidative stress related to various diseases, such as obesity and diabetes mellitus. The activity of the Nrf2-ARE (antioxidant response element) signaling was evaluated by a reporter assay involving over five hundred various edible medicinal herbs, and the highest Nrf2 activity was found in the ethanol extract of Peucedanum japonicum leaves. The active compound in the extract was isolated by high performance liquid chromatography (HPLC), and the chemical structure was identical to pteryxin based on ¹H, ¹³C-NMR spectra and liquid chromatography/time-of-fright mass spectrometer (LC/TOF/MS). From the pteryxin, the transcription factor Nrf2 was accumulated in the nucleus and resulted in the expression of the antioxidant protein, heme oxygenase-1 (HO-1). In addition, the Nrf2 activity involving HO-1 expression due to coumarin derivatives was evaluated together with pteryxin. This suggested that the electrophilicity, due to the α,β-carbonyl and/or substituted acyl groups in the molecule, modulates the cysteine residue in Keap1 via the Michel reaction, at which point the Nrf2 is dissociated from the Keap1. These results suggest that pteryxin will be a useful agent for developing functional foods.

Keywords: HO-1; Nrf2; Peucedanum japonicum Thunb; RAW264.7 cells; coumarin; oxidative stress; pteryxin.

Figure 1

Chemical structures of pteryxin and the derivatives used in this study.

Figure 2

Activation of the Nrf2 (Nuclear factor-erythroid-2-related factor)-ARE (antioxidant response element) signaling in the presence of the ethanol extract of Peucedanum japonicum Thunb leaves in RAW264.7 macrophage cells. (a) Cell viability with treated samples at the test concentrations was examined by an MTT assay. The cell viability was expressed as % of the control cells without sample. The Nrf2-ARE signaling activity of the EtOH extract of the P. japonicum leaves was evaluated by the reporter assay as described in the text. (b) The effect of the various concentrations (100–400 μg/mL) of the EtOH extract of P. japonicum leaves in RAW264.7 cells. The activity (%) was indicated as % of induction for the control cells without a sample. Data were expressed as mean ± SD, and the significant difference was analyzed by the student’s t-test. * p < 0.01 indicated as a significant difference from the control.

Figure 3

Nrf2-ARE signaling activity due to pteryxin in RAW264.7 macrophage cells. The Nrf2-ARE signaling activity in the presence of pteryxin (25–100 µM) was evaluated by the reporter assay, as described in the text. (a) Cell viability with treated samples at the test concentrations was examined by an MTT assay. The cell viability was expressed as % of control cells without a sample. (b) The effect of the various concentrations (25–100 µM) of pteryxin in RAW264.7 cells. The activity was indicated as induction (%) for control cells without sample. Data were expressed as mean ± SD, and the significant difference was analyzed by the student’s t-test. * p < 0.01 indicated a significant difference from the control.

Figure 4

Expression of the transcription factor Nrf2 protein due to pteryxin in RAW 264.7 macrophage cells. The Nrf2 protein expression in the presence of a compound was detected by western blot analysis and determined by densitometry. (a) Cytoplasmic Nrf2 protein expression and (b) nuclear Nrf2 protein expression in the presence of a compound. Data were expressed as mean ± SD, and the significant difference was analyzed by the student’s t-test. * p < 0.01 and ** p < 0.05 indicated a significant difference from the control.

Figure 5

HO-1 (heme oxygenase-1) expression due to pteryxin in RAW 264.7 macrophage cells. The HO-1 protein expression due to pteryxin (25–100 µM) on the Nrf2-ARE signaling in the cells was examined. (a) Western blot analysis of the HO-1 protein in the presence of a compound. (b) Densitometry analysis of the expression of the HO-1 protein. Data were expressed as mean ± SD, and the significant difference was analyzed by the student’s t-test. * p < 0.01 indicated a significant difference from the control.

Figure 6

Activation of Nrf2-ARE signaling and the HO-1 protein expression due to coumarin derivatives in the RAW 264.7 macrophage cells. (a) The activation of Nrf2-ARE signaling due to the coumarin derivatives (50 µM) was assessed by the reporter assay. (b) The HO-1 protein expression due to the various coumarins (50 µM) was detected by Western blot analysis. The expression of the HO-1 protein was determined by a densitometry analysis. Data were expressed as mean ± SD, and the significant difference was analyzed by the student’s t-test. * p < 0.01 indicated a significant difference from the control.