Anti-Inflammatory Activity of Acacia Honey through Inhibition of NF-κB and MAPK/ATF2 Signaling Pathway in LPS-Stimulated RAW264.7 Cells

Abstract

Honey used as conventional medicine has various pharmacological properties. In the honey and anti-inflammatory effect, Gelam honey and Manuka honey has been reported to exert anti-inflammatory activity. However, the anti-inflammatory effect and potential mechanisms of acacia honey (AH) are not well understood. In this study, we investigated anti-inflammatory activity and mechanism of action of AH in LPS-stimulated RAW264.7 cells. AH attenuated NO production through inhibition of iNOS expression in LPS-stimulated RAW264.7 cells. AH also decreased the expressions of $IL-1{\beta}$, IL-6 and $TNF-{\alpha}$ as pro-inflammatory cytokines, and MCP-1 expression as a pro-inflammatory chemokine. In the elucidation of the molecular mechanisms, AH decreased LPS-mediated $I{\kappa}B$-${\alpha}$ degradation and subsequent nuclear accumulation of p65, which resulted in the inhibition of $NF-{\kappa}B$ activation in RAW264.7 cells. AH dose-dependently suppressed LPS-mediated phosphorylation of ERK1/2 and p38 in RAW264.7 cells. In addition, AH significantly inhibited ATF2 phosphorylation and nuclear accumulation of ATF2 in LPS-stimulated RAW264.7 cells. These results suggest that AH has an anti-inflammatory effect, inhibiting the production of pro-inflammatory mediators such as NO, iNOS, $TNF-{\alpha}$, IL-6, $IL-1{\beta}$ and MCP-1 via interruption of the $NF-{\kappa}B$ and MAPK/ATF2 signaling pathways.

Fig. 1. AH attenuates NO production through inhibition of iNOS expression in LPS-stimulated RAW264.7 cells. (A) RAW264.7 cells were pretreated with the honey extracts according to honey plants for 6 h and then co-treated with LPS (1 ㎍/㎖) for 18 h. The determination of NO was measured by Griess assay. (B) RAW264.7 cells were pretreated with AH for 6 h and then co-treated with LPS (1㎍/㎖) for 18 h. The determination of NO was measured by Griess assay. (C) RAW264.7 cells were pretreated with AH for 6 h and then co-treated with LPS (1 ㎍/㎖) for 18 h. Total RNA was prepared after LPS and AH treatment. GAPDH was used as internal control for RT-PCR. (D) RAW264.7 cells were pretreated with AH for 24 h or 48 h. The cell viability was measured by MTT assay. ^# P < 0.05 compared to the cells without the treatment alone, and * P < 0.05 compared to the cells treated with LPS.

Fig. 2. AH inhibits the expression of pro-inflammatory mediators in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pretreated with AH for 6 h and then co-treated with LPS (1 ㎍/㎖) for 18 h. Total RNA was prepared after LPS and AH treatment. GAPDH was used as internal control for RT-PCR. ^#P < 0.05 compared to the cells without the treatment alone, and *P < 0.05 compared to the cells treated with LPS.

Fig. 3. AH inhibits NF-κB signaling in LPS-stimulated RAW264.7 cells. (A) RAW264.7 cells were pretreated with AH for 6 h and then co-treated with LPS (1 ㎍/㎖) for 30 min. (B) RAW264.7 cells were pretreated with AH for 6 h and then co-treated with LPS (1 ㎍/㎖) for 45 min. After the treatment, the nucleus was prepared. For Western blot analysis (A and B), the cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against IκB-α and p65. Actin and TBP were used as internal control for Western blot analysis. (C) RAW264.7 cells were co-transfected with NF-κB luciferase constructs and pRL-null. The cells were pretreated with AH for 6 h and then co-treated with LPS (1 ㎍/㎖) for 18 h. Luciferase activity for NF-κB was measured as a ratio of firefly luciferase signal/renilla luciferase signal using a dual luciferase assay kit. ^#P < 0.05 compared to the cells without the treatment alone, and * P < 0.05 compared to the cells treated with LPS.

Fig. 4. AH inhibits MAPK/ATF2 signaling in LPS-stimulated RAW264.7 cells. (A and B) RAW264.7 cells were pretreated with AH for 6 h and then co-treated with LPS (1 ㎍/㎖) for 30 min. (C) RAW264.7 cells were pretreated with AH for 6 h and then co-treated with LPS (1 ㎍/㎖) for 1 h. After the treatment, the nucleus was prepared. For Western blot analysis (A, B and C), the cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-ERK1/2, p-p38 and p-ATF2. Total-ERK1/2, total-p38, total-ATF2 and TBP were used as internal control for Western blot analysis.