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. 2023 Feb 9:6:100459.
doi: 10.1016/j.crfs.2023.100459. eCollection 2023.

Characterization of tangeretin as an activator of nuclear factor erythroid 2-related factor 2/antioxidant response element pathway in HEK293T cells

Chengyu Lv  1   2 , Yuqiu Li  2 , Rong Liang  3 , Wei Huang  2 , Yechen Xiao  1 , Xinqi Ma  2 , Yongjun Wang  2 , Haoyang Zou  1 , Fen Qin  2 , Chang Sun  2 , Tiezhu Li  2 , Jie Zhang  1
Affiliations

Characterization of tangeretin as an activator of nuclear factor erythroid 2-related factor 2/antioxidant response element pathway in HEK293T cells

Chengyu Lv et al. Curr Res Food Sci. .

Abstract

Numerous studies have reported that tangeretin is a polymethoxylated flavone with a variety of biological activates, but little research has been done on the antioxidant mechanism of tangeretin. Hence, we investigated the effect of tangeretin on the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway and its potential molecular mechanisms by in vitro and in silico research. The results of molecular docking suggested that tangeretin bound at the top of the central pore of Kelch-like ECH-associated protein 1 (Keap1) Kelch domain, and the hydrophobic and hydrogen bond interactions contributed to their stable binding. Herein, the regulation of Nrf2-ARE pathway by tangeretin was explored in the human embryonic kidney cell line HEK293T, which is relatively easy to be transfected. Upon binding to tangeretin, Nrf2 translocated to the nucleus of HEK293T cells, which in turn activated the Nrf2-ARE pathway. Luciferase reporter gene analysis showed that tangeretin significantly induced ARE-mediated transcriptional activation. Real-time PCR and Western blot assays showed that tangeretin induced the gene and protein expressions of Nrf2-mediated targets, including heme oxygenase 1 (HO-1), nicotinamide adenine dinucleotide phosphate (NADPH) quinone dehydrogenase 1 (NQO1), and glutamate-cysteine ligase (GCLM). In addition, tangeretin could effectively scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. In summary, tangeretin may be a potential antioxidant via activating the Nrf2-ARE pathway.

Keywords: Antioxidant response element; Molecular docking; Nuclear factor erythroid 2-related factor 2; Tangeretin.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Structures of tangeretin (5,6,7,8,4′-pentamethoxyflavone).
Fig. 2
Fig. 2
Computational alignment of re-docked ligand (S,R,S)-1a (cyan sticks) and co-crystallized ligand (S,R,S)-1a (magenta sticks) at the top of the central pore of Keap1 Kelch domain. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3
Fig. 3
The result of molecular docking between tangeretin (green sticks) and Keap1 Kelch domain. Co-crystallized ligand (S,R,S)-1a, magenta sticks; hydrogen bonds, red dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4
Fig. 4
Cytotoxicities of tangeretin (TAN) (A) and H2O2 (B). Results are given as means ± SD of three independent experiments and normalized to DMSO control group. *, **, ***, statistically significant differences (p < 0.05, p < 0.01, p < 0.001).
Fig. 5
Fig. 5
Effect of tangeretin on nuclear translocation of Nrf2. (A) The expression levels of nuclear Nrf2 and cytosolic Nrf2 in HEK293T cells were detected by Western blot after treatment with tangeretin for 18 h, followed by 500 μM H2O2 for 8 h. (B) The levels of nuclear Nrf2 were quantified by densitometry. (C) The levels of cytosolic Nrf2 were quantified by densitometry. (D) The levels of nuclear Nrf2/cytosolic Nrf2 were quantified by densitometry. Each group of experiments was determined three times and given as mean ± SD. **, ***, statistically significant differences (p < 0.01 and p < 0.001).
Fig. 6
Fig. 6
HEK293T cells were treated with tangeretin or tert-butylhydroquinone (t-BHQ) for 24 h, and then the luciferase activity was measured. Results are given as means ± SD of three independent experiments and normalized to DMSO control group. **, ***, statistically significant differences (p < 0.01 and p < 0.001).
Fig. 7
Fig. 7
Effect of tangeretin on the expression of Nrf2-mediated target gene. Tangeretin was used for 18 h, followed by 500 μM H2O2 for 8 h. (A) (B) and (C) are the relative mRNA levels of HO-1, NQO1 and GCLM in HEK293T cells, respectively. Results are given as means ± SD of three independent experiments and normalized to H2O2 control group. *, **, ***, statistically significant differences (p < 0.05, p < 0.01 and p < 0.001, respectively).
Fig. 8
Fig. 8
Effect of tangeretin on the protein levels of HO-1, NQO1 and GCLM. (A) The expression levels of HO-1, NQO1 and GCLM in HEK293T cells were detected by Western blot after treatment with tangeretin for 18 h, followed by 500 μM H2O2 for 8 h. (B) The levels of HO-1 were quantified by densitometry. (C) The levels of NQO1 were quantified by densitometry. (D) The levels of GCLM were quantified by densitometry. Each group of experiments was determined three times and given as mean ± SD. *, ***, statistically significant differences (p < 0.05 and p < 0.001).
Fig. 9
Fig. 9
Scavenging effect of tangeretin and tert-butylhydroquinone on DPPH free radicals. Results are given as means ± SD of three independent experiments and normalized to DMSO control group. **, ***, statistically significant differences (p < 0.01 and p < 0.001).
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