Psoralen and Isopsoralen Activate Nuclear Factor Erythroid 2-Related Factor 2 Through Interaction With Kelch-Like ECH-Associated Protein 1

Abstract

As natural furocoumarins, psoralen and its isomer isopsoralen are widely distributed in various fruits including Ficus carica L., vegetables including celery, and medicinal herbs including Psoralea corylifolia L. Although psoralen and isopsoralen have been used as dietary supplements because of their bioactivities such as antibacterial and anti-inflammatory properties; however, the potential mechanisms underlying the antioxidant activities of these two furocoumarins still need to be explored. Hence, the aims of this work were to examine the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) by psoralen and isopsoralen, as well as the binding interaction of Kelch-like ECH-associated protein 1 (Keap1) with these two furocoumarins. Interestingly, both psoralen and isopsoralen induced Nrf2 nuclear translocation in a dose-dependent manner in HEK293T cells. These two furanocoumarins also activated antioxidant response element (ARE)-driven luciferase activity. The mRNA expression of GCLM, HO-1, and NQO1 genes was significantly upregulated by treatment of HEK293T cells with psoralen and isopsoralen, respectively. Similarly, the expression of proteins can be promoted. Both psoralen and isopsoralen were located in the top of the central pocket of the Keap1 Kelch domain, suggesting that they were natural ligands of Keap1. In conclusion, both psoralen and isopsoralen activate Nrf2 through interaction with Keap1, thereby serving as natural antioxidants.

Keywords: Nrf2; antioxidant response element; isopsoralen; molecular docking; psoralen.

FIGURE 1

The structures of psoralen (A) and isopsoralen (B).

FIGURE 2

Free radical scavenging ability of psoralen and isopsoralen. (A) DPPH radical scavenging ability. (B) ABTS radical scavenging ability. (C) Hydroxyl radical scavenging ability.

FIGURE 3

Cytotoxicities of psoralen (A), isopsoralen (B), and H₂O₂ (C) on HEK293T cells. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the DMSO groups.

FIGURE 4

Western blot analysis of the effects of psoralen and isopsoralen on the expression of Nrf2 nuclear and Nrf2 cytoplasmic proteins. Expression levels of nuclear Nrf2 protein and cytoplasmic Nrf2 protein in HEK293T cells treated with (A) psoralen and (B) isopsoralen. The levels of (C) psoralen nuclear Nrf2 and (D) isopsoralen nuclear Nrf2 were quantified by densitometry. The levels of (E) psoralen cytosolic Nrf2and (F) isopsoralen cytosolic Nrf2 were quantified by densitometry. *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 5

ARE luciferase activity of psoralen and isopsoralen was measured by the luciferase reporter assay. DMSO and tert‐butylhydroquinone (t‐BHQ) (10 μM) were used as negative and positive controls. ^*/#, ^##/$$, and ^***/$$$ represent statistically significant differences of p < 0.05, p < 0.01, and p < 0.0001, respectively.

FIGURE 6

Real‐time quantitative PCR analysis of the effects of psoralen (A) and isopsoralen (B) on GCLM, HO‐1, and NQO1 gene expression. The color scale represents relative mRNA levels.

FIGURE 7

Western blotting analysis of the effects of psoralen and isopsoralen on the expression of GCLM, HO‐1, and NQO1 proteins. Expression levels of GCLM, HO‐1, and NQO1 proteins in HEK293T cells treated with (A) psoralen and (B) isopsoralen. (C) The levels of GCLM by (C) psoralen and (D) isopsoralen were quantified by densitometry. The levels of HO‐1 by (E) psoralen and (F) isopsoralen were quantified by densitometry. The levels of NQO1 by (G) psoralen and (H) isopsoralen were quantified by densitometry. **p < 0.01 and ***p < 0.001.

FIGURE 8

The binding mode of psoralen (A, C) and isopsoralen (B, D) in the binding pocket of the Keap1 Kelch domain. Hydrogen bonds are shown as red dotted lines.