Farnesoid X receptor agonists attenuate colonic epithelial secretory function and prevent experimental diarrhoea in vivo.

OBJECTIVE: Bile acids are important regulators of intestinal physiology, and the nuclear bile acid receptor, farnesoid X receptor (FXR), is emerging as a promising therapeutic target for several intestinal disorders. Here, we investigated a role for FXR in regulating intestinal fluid and electrolyte transport and the potential for FXR agonists in treating diarrhoeal diseases. DESIGN: Electrogenic ion transport was measured as changes in short-circuit current across voltage-clamped T84 cell monolayers or mouse tissues in Ussing chambers. NHE3 activity was measured as BCECF fluorescence in Caco-2 cells. Protein expression was measured by immunoblotting and cell surface biotinylation. Antidiarrhoeal efficacy of GW4064 was assessed using two in vivo mouse models: the ovalbumin-induced diarrhoea model and cholera toxin (CTX)-induced intestinal fluid accumulation. RESULTS: GW4064 (5 μmol/L; 24 h), a specific FXR agonist, induced nuclear translocation of the receptor in T84 cells and attenuated Cl(-) secretory responses to both Ca(2+) and cAMP-dependent agonists. GW4064 also prevented agonist-induced inhibition of NHE3 in Caco-2 cells. In mice, intraperitoneal administration of GW4064 (50 mg/mL) also inhibited Ca(2+) and cAMP-dependent secretory responses across ex vivo colonic tissues and prevented ovalbumin-induced diarrhoea and CTX-induced intestinal fluid accumulation in vivo. At the molecular level, FXR activation attenuated apical Cl(-) currents by inhibiting expression of cystic fibrosis transmembrane conductance regulator channels and inhibited basolateral Na(+)/K(+)-ATPase activity without altering expression of the protein. CONCLUSIONS: These data reveal a novel antisecretory role for the FXR in colonic epithelial cells and suggest that FXR agonists have excellent potential for development as a new class of antidiarrheal drugs.</p

Chemical structure of the most potent antioxidant rotenoids.

<p>Rotenoids were obtained from Kupchan partitioning of the methanol extract of <i>B. diffusa</i> root following by sequential silica gel column chromatography and HPLC.</p

Effect of boeravinone G (0.1–1 ng/ml) on Fenton's reagent (H₂O₂/Fe²⁺ 1 mM)-induced malondialdehyde-equivalents (MDA-equivalents) production.

<p>Effect observed in differentiated Caco-2 cells after 24-hour boeravinone G exposure. Data represent mean ± SEM of 6 experiments. ^#p<0.001 <i>vs</i> control (vehicle) and ***p<0.001 <i>vs</i> H₂O₂/Fe²⁺ alone.</p

Effect of boeravinone G (0.1–1 ng/ml) on pERK₁ (A) and pERK₂ (B) expression.

<p>Quantitative analysis and representative western blot analysis of pERK₁ and pERK₂ in Caco-2 cells exposed to Fenton's reagent (H₂O₂/Fe²⁺ 1 mM) without or with boeravinone G (0.1–1 ng/ml). The results were normalized with anti-ERK₂ (pERK_1/2/ERK₂). ^#p<0.01 <i>vs</i> control (vehicle); ***p<0.001 <i>vs</i> H₂O₂/Fe²⁺ alone.</p

Effect of boeravinone G (0.1–1 ng/ml) on Fenton's reagent (H₂O₂/Fe²⁺ 2 mM)-induced reactive species (ROS) production.

<p>Effect observed in differentiated Caco-2 cells after 24-hour boeravinone G exposure. Data represent mean ± SEM of 6 experiments. ^#p<0.001 <i>vs</i> control (vehicle); *p<0.05, **p<0.01 and ***p<0.001 <i>vs</i> H₂O₂/Fe²⁺ alone.</p

Effect of boeravinone G (0.1–1 ng/ml) on superoxide dismutase (SOD) activity.

<p>SOD activity was evaluated in Caco-2 cells exposed to Fenton's reagent (H₂O₂/Fe²⁺ 1 mM) without or with boeravinone G (0.1–1 ng/ml). Data represent mean ± SEM of 4 experiments. ^#p<0.001 <i>vs</i> control (vehicle); *p<0.05 and ***p<0.001 <i>vs</i> H₂O₂/Fe²⁺ alone; °p<0.05 <i>vs</i> control.</p

Effect of boeravinone G (BG, 0.1–1 ng/ml) on DNA damage.

<p>DNA damage (tail intensity) was detected by the Comet assay in Caco-2 cells exposed to 75 µM H₂O₂ for 5 min in absence or presence of boeravinone G. a = control; b = H₂O₂ 75 µM; c = H₂O₂ 75 µM+BG 0.1 ng/ml; d = H₂O₂ 75 µM+BG 0.3 ng/ml; e = H₂O₂ 75 µM+BG 1 ng/ml. Data represent mean ± SEM of 4 experiments. ^#p<0.001 <i>vs</i> control (vehicle) and ***p<0.001 <i>vs</i> H₂O₂ alone.</p

Antioxidant activity (AA), detected using ESR assay, of the fractions obtained from the carbon tetrachloride extract of <i>B. diffusa</i> root.

<p>Antioxidant activity (AA), detected using ESR assay, of the fractions obtained from the carbon tetrachloride extract of <i>B. diffusa</i> root.</p

Effect of 5 mg/ml of <i>Boerhaavia diffusa</i> methanol extract on <i>electron spin resonance spectroscopy</i>.

<p>Representative ESR spectra of DMPO-OH spin adduct signal (A) and DMPO-OH spin adduct signal in the presence of 5 mg/ml of <i>Boerhaavia diffusa</i> methanol extract (B).</p