Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Abstract

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

Keywords: FoxO3a/autophagy signaling pathway; inflammatory responses; punicalagin.

Figure 1

Cell viability of PU and LPS on RAW264.7 macrophages. (A–B) RAW264.7 cells were treated with various concentrations of PU and LPS for 24 h and 48 h. The cell viability was determined by MTT assay, as described in Materials and Methods. The data are presented as means ± SD (n = 3). (**, p < 0.01 vs. control group).

Figure 2

Photograph of RAW264.7 cells were pre-treated with various concentration of PU for 1 h and treated with LPS for an additional 24 h under optical microscopy (magnification x400). (A) Control; (B) LPS treatment; (C–E) LPS and PU (12.5, 25, and 50 µM) treatment; (F) PU treatment; and (G) cell viability of PU supplementation with LPS-induced RAW264.7 macrophages. The RAW264.7 cells were pre-treated with various concentrations of PU for 1 h and LPS for an additional 24 h. The cell viability was determined by MTT assay, as described in Materials and Methods. The data are presented as means ± SD (n = 3). (**, p < 0.01 vs. control group).

Figure 3

Anti-inflammatory effect of PU on LPS-induced RAW264.7 macrophages. (A–C) Cells were pre-treated with various concentrations of PU for 1 h and treated with LPS for an additional 24 h. The NO content was determined by Griess reagent and the production of cytokines were measured by enzyme-linked immunosorbent assay (ELISA) kit using the microplate reader. The data are presented as means ± SD (n = 3). (*, p < 0.05 and ** p < 0.01 vs. LPS group, and ##, p < 0.001 vs. control group).

Figure 4

Inhibitive effects of PU on LPS-induced p65 (A), JNK (B), ERK1/2 (C) and p38 (D) phosphorylation in RAW264.7 macrophages. Cells were pre-treated with PU (12.5–50 μM) for 1 h before exposure to LPS (1 μg/mL) for 30 minutes. The data are presented as means ± SD (n = 3). (*, p < 0.05 and ** p < 0.01 vs. LPS group, and ##, p < 0.01 vs. control group).

Figure 5

Effect of PU on autophagy inhibition in RAW 264.7 macrophages. (A–C) Cells were pre-treated with various concentrations of PU (12.5–50 μM) for 1 h and treated with LPS (1 μg/mL) for an additional 24 h. LC3II (A), p62 (B), Beclin1 (C) expression levels were determined by Western blot analysis. Autophagolysosomes were determined by MDC assays (D) under a fluorescence microscope (magnification x200). Representative image from three independent experiments has been shown along with β-actin as internal loading control. (E and F) Cells were pre-treated with chloroquine (20 μM) for 2 h, followed by treatment with PU (50 μM) for 1 h and treated with LPS (1 μg/mL) for an additional 24 h. LC3II (E), p62 (F) protein level were examined by Western blot analysis as described previously. Autophagolysosomes was determined by MDC assays (G) under a fluorescence microscope (magnification x200). Images are representative of three independent experiments that showed similar results. The data are presented as means ± SD (n = 3). (**, p < 0.01 vs. LPS group; ##, p < 0.01 vs. control group; &&, p < 0.01 vs. control with CQ treatment group; $$, p < 0.01 vs. LPS with LPS group).

Figure 5

Effect of PU on autophagy inhibition in RAW 264.7 macrophages. (A–C) Cells were pre-treated with various concentrations of PU (12.5–50 μM) for 1 h and treated with LPS (1 μg/mL) for an additional 24 h. LC3II (A), p62 (B), Beclin1 (C) expression levels were determined by Western blot analysis. Autophagolysosomes were determined by MDC assays (D) under a fluorescence microscope (magnification x200). Representative image from three independent experiments has been shown along with β-actin as internal loading control. (E and F) Cells were pre-treated with chloroquine (20 μM) for 2 h, followed by treatment with PU (50 μM) for 1 h and treated with LPS (1 μg/mL) for an additional 24 h. LC3II (E), p62 (F) protein level were examined by Western blot analysis as described previously. Autophagolysosomes was determined by MDC assays (G) under a fluorescence microscope (magnification x200). Images are representative of three independent experiments that showed similar results. The data are presented as means ± SD (n = 3). (**, p < 0.01 vs. LPS group; ##, p < 0.01 vs. control group; &&, p < 0.01 vs. control with CQ treatment group; $$, p < 0.01 vs. LPS with LPS group).

Figure 6

Anti-inflammatory effect of PU on LPS-induced RAW264.7 macrophages. (A–C) Cells were pre-treated with chloroquine (20 μM) for 2 h, followed by treatment with PU (50 μM) for 1 h and treated with LPS (1 μg/mL) for an additional 24 h. The NO content was determined by Griess reagent and the production of cytokines were measured by enzyme-linked immunosorbent assay (ELISA) kit using the microplate reader. The data are presented as means ± SD (n = 3). (*, p < 0.05 and **, p < 0.01 vs. LPS group; #, p < 0.05 and ##, p < 0.01 vs. control group; $, p < 0.05 and $$, p < 0.01 vs. LPS combined with CQ group).

Figure 7

Effects of PU (A) on LPS-induced Foxo3a in RAW264.7 macrophages. Cells were pre-treated with PU (12.5–50 μM) for 1 h before exposure to LPS (1 μg/mL) for 30 minutes. Representative image from three independent experiments are shown along with β-actin as the internal loading control. The data are presented as means ± SD (n = 3). (**, p < 0.01 vs. LPS group; and ##, p < 0.01 vs. control group).