Amaranth's 2-Caffeoylisocitric Acid-An Anti-Inflammatory Caffeic Acid Derivative That Impairs NF-κB Signaling in LPS-Challenged RAW 264.7 Macrophages

Abstract

For centuries, Amaranthus sp. were used as food, ornamentals, and medication. Molecular mechanisms, explaining the health beneficial properties of amaranth, are not yet understood, but have been attributed to secondary metabolites, such as phenolic compounds. One of the most abundant phenolic compounds in amaranth leaves is 2-caffeoylisocitric acid (C-IA) and regarding food occurrence, C-IA is exclusively found in various amaranth species. In the present study, the anti-inflammatory activity of C-IA, chlorogenic acid, and caffeic acid in LPS-challenged macrophages (RAW 264.7) has been investigated and cellular contents of the caffeic acid derivatives (CADs) were quantified in the cells and media. The CADs were quantified in the cell lysates in nanomolar concentrations, indicating a cellular uptake. Treatment of LPS-challenged RAW 264.7 cells with 10 µM of CADs counteracted the LPS effects and led to significantly lower mRNA and protein levels of inducible nitric oxide synthase, tumor necrosis factor alpha, and interleukin 6, by directly decreasing the translocation of the nuclear factor κB/Rel-like containing protein 65 into the nucleus. This work provides new insights into the molecular mechanisms that attribute to amaranth's anti-inflammatory properties and highlights C-IA's potential as a health-beneficial compound for future research.

Keywords: NF-κB; RAW 264.7 macrophages; amaranth; caffeic acid derivatives; inflammation.

Figure 1

(A) Concentrations of CADs in the RAW264.7 cell supernatants after 4 h and 24 h incubation with 10 µM solutions and a basal concentration of CA, CQA, and C-IA in cell culture medium (RPMI 1640) (n = 3); (B) Lysate concentrations of CA, CQA, and C-IA in whole lysates of RAW264.7 cells after 4 h incubation with 10 µM solutions and the control (0.1% EtOH) (n = 3); (C) Lysate concentrations of CA, CQA, and C-IA in whole lysates of RAW264.7 macrophages after 24 h incubation with 10 µM solutions and the control (0.1% EtOH) (n = 3); Statistically significant differences derived from one-way ANOVA Tukey’s post-hoc test are indicated by * (p ≤ 0.05 vs. control).

Figure 2

(A) iNOS mRNA expression in RAW 264.7 cells after 16 h of incubation with 10 µM CA, CQA, and C-IA solutions (n = 3–4); (B) iNOS protein levels in RAW 264.7 cells after 24 h incubation with 10 µM CA, CQA, and C-IA solutions (n = 3); (C) NO concentrations in the supernatants of RAW 264.7 cells in nmol/mg protein after 24 h incubation with 10 µM CA, CQA, and C-IA solutions (n = 3); Statistically significant differences derived from one-way ANOVA Tukey’s post-hoc test are indicated by * (p ≤ 0.05 vs. control) or # (p ≤ 0.05 vs. LPS). Representative blots are depicted in Supplemental Figure S2A.

Figure 3

(A) p65 protein levels in the nuclear fraction of RAW 264.7 cells after 4 h incubation with 10 µM CA, CQA, and C-IA solutions (n = 3); (B) p65 protein levels in the cytosolic fraction of RAW 264.7 cells after 4 h incubation with 10 µM CA, CQA, and C-IA solutions (n = 4); Statistically significant differences derived from one-way ANOVA Tukey’s post-hoc test are indicated by * (p ≤ 0.05 vs. control) or # (p ≤ 0.05 vs. LPS). Representative blots are depicted in Supplemental Figure S2B/C.

Figure 4

(A) TNFα mRNA expression in RAW 264.7 cells after 4 h incubation with 10 µM CA, CQA, and C-IA solutions (n = 3–4); (B) TNFα protein in the supernatant of RAW 264.7 cells in pg/mg protein after 24 h incubation with 10 µM CA, CQA, and C-IA solutions n = 3); (C) IL-6 mRNA expression in RAW 264.7 cells after 4 h incubation with 10 µM CA, CQA, and C-IA solutions (n = 3); (D) IL-6 protein in the supernatant of RAW 264.7 cells after 24 h incubation with 10 µM CA, CQA, and C-IA solutions (n = 3). Statistically significant differences, derived from one-way ANOVA Tukey’s post-hoc test are indicated by * (p ≤ 0.05 vs. control) or # (p ≤ 0.05 vs. LPS).