The effect of a fennel seed extract on the STAT signaling and intestinal barrier function

Abstract

Background: Foeniculum vulgare, F. vulgare, commonly known as fennel, is believed to be one of the world's oldest medicinal herbs and has been exploited by people for centuries as a nutritional aid for digestive disorders. In many southeast Asian countries, it is ingested as an after-meal snack, mukhvas, due to its breath-freshening and digestive aid properties. F. vulgare is used in some countries, such as Iran, as a complementary and alternative treatment for inflammatory bowel disease (IBD).

Methods: This study investigated the effects of fennel seed extract on intestinal epithelium barrier function and the Signal Transducer and Activator of Transcription (STAT) pathway. This pathway is active in inflammatory bowel disease. To study the protective effects of fennel seed extract in vitro, monolayers derived from the T84 colonic cell line were challenged with interferon-gamma (IFN-γ) and monitored with and without fennel seed extract. To complement our in vitro studies, the dextran sodium sulfate induced murine colitis model was employed to ascertain whether the protective effect of fennel seed extract can be recapitulated in vivo.

Results: Fennel seed extract was shown to exert a protective effect on transepithelial electrical resistance (TEER) in both T84 and murine models and showed increases in tight junction-associated mRNA in T84 cell monolayers. Both models demonstrated significant decreases in phosphorylated STAT1 (pSTAT1), indicating reduced activation of the STAT pathway. Additionally, mice treated with fennel seed showed significantly lower ulcer indices than control mice.

Conclusions: We conclude barrier function of the gastrointestinal tract is improved by fennel seed extract, suggesting the potential utility of this agent as an alternative or adjunctive therapy in IBD.

Fig 1. Fennel seed extract (FN) treatment of T84 cells improves tight junction functionality.

A) Fennel seed extract (FN) pretreatment (with increasing dosage) of T84 cells treated with IFN-γ prevents decrease of TEER. B)) Fennel seed extract (FN) addition post IFN-γ treatment shows restorative effects on TEER. C) Propidium Iodide staining of Control, FN, and Vehicle treated T84 cells show no change in cell viability. D) Fennel seed extract (FN) treatments increase transcription of tight junction genes TJP-1 and OCLD. N = 4; a = P < 0.05; b = P < 0.01; c = P < 0.001; Data shown as mean + SD. Significance calculated against IFN-γ group in all experiments.

Fig 2

Fennel seed extract (FN) treatment (7.5 μl/ml) prevents the mislocalization and disruption of Tight junction protein 1 (TJP-1) (A) and Occludin (B) in IFN-γ treated T84 cells. Scale bar represents 50 μm. Representative confocal images from at least 5 independent experiments.

Fig 3. Fennel seed extract (FN) treated T84 cells show decreased STAT1 activation.

Western blot analysis of pSTAT1 and STAT1 show decreasing pSTAT1 with increasing doses of fennel seed extract. N = 3; a = P < 0.05; b = P < 0.01; c = P < 0.001; FN 1-FN 4 correlate to increasing doses of fennel seed extract. Data shown as mean + SD. Significance calculated against IFN-γ group in all experiments.

Fig 4. Fennel seed extract has beneficial effects on barrier integrity in mice with DSS colitis.

A) fennel seed extract (FN) prevents loss of TEER in FN/DSS treated mice compared to DSS treated mice. B) fennel seed extract (FN) treated mice show improved ulcer indices compared to DSS treated mice. Inflammation sub score also shown, mirror improvement in overall ulcer indices. C) Representative H&E staining of mid colons of treated mice showing reduced inflammation, ulceration and architectural changes in fennel seed extract (FN) treated mice. D) Representative H&E staining of mid colon demonstrating inflammation in DSS and Fennel seed extract/DSS treated mice. N = 4; Fig 3C: 20x magnification, Fig 3D: 10x magnification; a = P < 0.05; c = P < 0.001.

Fig 5. Fennel seed extract reduces protein expression of pSTAT1 in mice with DSS colitis.

Western blot analysis demonstrates significantly reduced pSTAT1 in FN/DSS mice compared to DSS mice. Quantification of band density shown. pSTAT1 and STAT1 density adjusted for β-actin levels. Data presented as mean + upper SD. N = 4; a = P < 0.05; c = P < 0.001.

Fig 6. Schematic representation of mechanism of STAT pathway attenuation in T84 model.

Fennel seed extract prevents phosphorylation of STAT1, preventing transcription of other inflammatory genes.