Anti-inflammatory Effect of Leaves Extracts from Aralia cordata through Inhibition of NF-κB and MAPKs Signaling in LPS-stimulated RAW264.7 Cells

Abstract

Aralia cordata (A. cordata), which belongs to Araliaceae, is a perennial herb widely distributed in East Asia. We evaluated the anti-inflammatory effect of stems (AC-S), roots (AC-R) and leaves (AC-L) extracted with 100% methanol of A. cordata and elucidated the potential signaling pathway in LPS-stimulated RAW264.7 cells. The AC-L showed a strong anti-inflammatory activity through inhibition of NO production. AC-L dose-dependently inhibited NO production by suppressing iNOS, COX-2 and $IL-{\beta}$ expression in LPS-stimulated RAW264.7 cells. AC-L inhibited the degradation and phosphorylation of $I{\kappa}B-{\alpha}$, which donated to the inhibition of p65 nuclear accumulation and $NF-{\kappa}B$ activation. Furthermore, AC-L suppressed the phosphorylation of ERK1/2 and p38. These results suggested that AC-L may utilize anti-inflammatory activity by blocking $NF-{\kappa}B$ and MAPK signaling pathway and indicated that the AC-L can be used as a natural anti-inflammatory drugs.

Fig. 1. Effect of the part of A. cordata on NO production in LPS-stimulated RAW264.7 cells. (A) RAW264.7 cells were pretreated with AC-S, AC-R and AC-L for 6 hours and then co-treated with LPS (1 ㎍/㎖) for 18 hours. The determination of NO production was measured by Griess assay. (B) RAW264.7 cells were treated with AC-L at the indicated concentrations for 24 hours. Cell viability was measured using MTT assay system and expressed as % cell viability. *P < 0.05 compared to the cells without the treatment, and #P < 0.05 compared to the cells treated with LPS alone.

Fig. 2. The effect of AC-L on NO production and iNOS, COX-2 and IL-1β expression in LPS-stimulated RAW264.7 cells. (A) RAW264.7 cells were pre-treated with AC-L at the indicated concentrations for 6 hours and then co-treated with LPS (1 ㎍/㎖) for the additional 18 hours. After treatment, NO production was measured using the media and Griess reagent and (B) cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and probed with iNOS and COX-2 antibody for Western blot. iNOS and COX-2 protein was visualized using ECL was used as a vehicle. (C) For RT-PCR, RAW264.7 cells were pre-treated with AC-L at the indicated concentrations for 6 hours and then co-treated with LPS (1 ㎍/㎖) for the additional 18 hours. Total RNA was isolated and RT-PCR was performed for iNOS and COX-2 and IL-1â. Values given are the mean ± SD (n = 3). *P < 0.05 compared to LPS treatment without AC-L. GAPDH was used as an internal control for RT-PCR.

Fig. 3. The effect of AC-L on NF-κB signaling activation in LPS-stimulated RAW264.7 cells. (A) RAW264.7 cells were pretreated with AC-L for 6 hours and then co-treated with LPS (1 ㎍/㎖) for 20 minutes. (B) RAW264.7 cells were pretreated with AC-L for 6 hours and then co-treated with LPS (1 ㎍/㎖) for 30 minutes. After the treatment, the cytosol and nucleus were prepared. For Western blot analysis, the cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against Iκ B-α and p65. Actin was used as internal control.

Fig. 4. The effect of AC-L on MAPK signaling activation in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pretreated with AC-L for 6 hours and then co-treated with LPS (1 ㎍/㎖) for 30 minutes. For Western blot analysis, the cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-ERK1/2, p-p38 and Total p-38 and Total ERK1/2 were used as internal control.