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. 2022 Nov 12;11(22):3607.
doi: 10.3390/foods11223607.

In Vitro Antioxidant Activity of Areca Nut Polyphenol Extracts on RAW264.7 Cells

Shuhan Yi  1 , Luyan Zou  2 , Zongjun Li  2   3   4 , Kozue Sakao  1   5 , Yuanliang Wang  2   3   4 , De-Xing Hou  1   5
Affiliations

In Vitro Antioxidant Activity of Areca Nut Polyphenol Extracts on RAW264.7 Cells

Shuhan Yi et al. Foods. .

Abstract

Chewing areca nuts is a popular hobby in the Asian region, and areca nuts are rich in polyphenols, although some alkaloids are included. In this study, we explored the antioxidant activity of areca nut polyphenols (ANP) in lipopolysaccharides (LPS)-stimulated RAW264.7 cells. The results revealed that ANP reduced the level of reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells and enhanced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). RNA-seq analysis showed that ANP down-regulated the transcription of genes related to the cancer pathway at 160 μg/mL, and the inflammatory pathway as well as viral infection pathway at 320 μg/mL. The cellular signaling analysis further revealed that the expressions of these genes were regulated by the mitogen-activated protein kinase (MAPK) pathway, and ANP downregulated the activation of the MAPK signaling pathway stimulated by LPS. Collectively, our findings showed that ANP inhibited the MAPK pathway and activated the Nrf2/HO-1 antioxidant pathways to reduce ROS generation induced by LPS.

Keywords: HO-1; Nrf2; RNA-seq; ROS; areca nut polyphenol.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Inhibitory effect of ANP on the intracellular ROS generation. The results shown are expressed as means ± SD (n = 3). The different letters in the graph indicated significance (p < 0.05).
Figure 2
Figure 2
Observation of cellular ROS by fluorescence inverted microscope.
Figure 3
Figure 3
ANP enhancement of the Nrf2 and HO-1 proteins: (a) Nrf2 and HO-1 were detected by Western blot and GAPDH was used as a control; (b,c) the densitometric quantification of bands showing Nrf2 and HO-1. The results shown are presented as means ± SD (n = 3). The different letters in the graph indicate significance (p < 0.05).
Figure 4
Figure 4
The differentially expressed genes volcano plots of 160/LPS (a) and 320/LPS (b). The padj is a p-value after correction for multiple hypothesis testing.
Figure 5
Figure 5
The GO analysis of all DEGs (a) gene ratio in 160/LPS, (b) gene ratio in 320/LPS, (c) number of genes in 160/LPS, (d) number of genes in 320/LPS. The padj is a p-value after correction for multiple hypothesis testing. The gene ratio is a ratio of the number of differential genes annotated to the GO term to the total number of differential genes.
Figure 6
Figure 6
The protein-protein interaction subnetwork of all DEGs network in 160/LPS.
Figure 7
Figure 7
The protein-protein interaction subnetwork of all DEGs network in 320/LPS.
Figure 8
Figure 8
ANP inhibited the phosphorylation of MEK1, ERK, MKK3, p38, MKK4, and JNK induced by LPS. Left pictures are western blots of indicated proteins and α-tubulin as a control. Right pictures are the density histogram of western blots. The “p-” means “phosphorylated”. The results shown are presented as means ± SD (n = 3). The different letters in the graph indicate significance (p < 0.05).
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