Freeze-dried water extract of avocado peel targets NF-kB and p38 MAPK activation to produce anti-inflammatory activity in human intestinal epithelial (HIEC-6) cells

Introduction

The dysregulation of the intestinal epithelium plays a crucial role in the development and progression of intestinal bowel diseases. This dysregulation is driven by cytokine production and activation of pro-inflammatory signalling pathways. Earlier studies on freeze-dried water extract of avocado peel revealed anti-inflammatory activity through reduction of TNF-α, IL-6 and IL-8 production in TNF-α-stimulated human intestinal epithelial (HIEC-6) cells [1]. Consequently, the present study explored the potential molecular mechanisms involved in the anti-inflammatory activity of freeze-dried water extract of avocado peel (FWEAP) in HIEC-6.

Method

Cells were treated with FWEAP (25, 50 and 100 μg/ml) for 60 min. Thereafter, they were stimulated with TNF-α (10 ng/ml). Protein level of phospho-p65 was measured using western blotting, while nuclear NF-κB p65 was detected using immunofluorescence microscopy. Transcriptional activity of NF-κB was evaluated using the luciferase reporter gene assay. Protein expression of phospho-p38 MAPK was also evaluated with western blotting. Data were analysed using one-way analysis of variance with post-hoc Dunnett's test (multiple comparisons).

Results

Stimulation of HIEC-6 cells with TNF-α (10 ng/ml) resulted in significant increase in protein levels of phospho-p65, nuclear NF-κB p65 and transcriptional activity, when compared with unstimulated cells. There was also a significant increase in protein level of phospho-p38 in comparison with unstimulated cells. Pre-treatment of HIEC-6 cells with FWEAP (25, 50 and 100 μg/ml) did not inhibit p65 phosphorylation in the cytoplasm. However, FWEAP suppressed nuclear levels of NF-κB p65 and significantly (p < 0.05) reduced NF-κB p65 transcriptional activity in a concentration dependent manner compared to stimulated cells. There was also a significant (p < 0.05) reduction in p-p38 MAPK when cells were treated with 50 and 100 μg/ml of FWEAP, when compared to stimulated cells.

Conclusions

These findings suggest that modulation of NF-κB and MAPK activation may be contributing to the anti-inflammatory activity of freeze-dried water extract of avocado peel in HIEC-6 cells.