ABSTRACT
1Scientific Institute of Gastroenterology “S de Bellis”, Castellana Grotte, Bari; 2 Dept. of General and Environmental Physiology, University of Bari.
In ex vivo and in vitro models olive oil phenols, including hydroxytyrosol (HT), have shown to have antioxidant properties especially on the vascular endothelium by decreasing the expression of cell adhesion molecules, increasing nitric oxide (NO) production and inducible NO synthesis [1, 2]. Nothing is known about HT participation on the intrinsic innervation of human colonic muscle mediated by nitric oxideand its effects on human colonic mucosa transport process. Therefore, we tested the effects of HT and L-arginine methyl ester (L-NAME), an inhibitor of NOS syntetasis, on electrically evoked contractions in human colonic circular muscle. Moreover we tested the effects of HT on electrophysiological parameters of stimulated human colon. Methods: Segments of sigmoid colon were obtained from 8 patients (mean age yrs, range 75-81 yrs), undergoing left hemicolectomy for non-obstructive sigmoid cancer. Colonic circular muscle strips and the corresponding mucosa were taken from macroscopically normal areas. Motility studies - Strips (10x3mm) deprived of the mucosal layer were mounted isometrically with a tension of 20-24mN in an organ bath with oxygenated in Krebs solution at 37° C. After stabilization of 60 min and recording of at least two comparable responses to carbachol (CARB, 100µM), the strips were exposed to HT and L-NAME. Moreover strips were exposed every 2 min to electrical field stimulation (EFS) delivering 10-sec pulses trains (0.1-10 Hz, duration 0.3 ms, 20V). The following drugs were tested: the association of atropine (ATR) (2µ M) and guanethidine (GUA) (5µ M), (30’ contact time); the association of ATR (2µ M), GUA (5µ M), HT (10µ M; 200µM) or L-NAME (200µM) (45’contact time) and the association of ATR (2µM), GUA (5µM), HT (10µM; 200µM) and L-NAME (200µM) (45’contact time). Electrically evoked contractions were also evaluated in the presence of tetrodotoxin (TTX, 1µM) (30 min contact time) to confirm that EFS-evoked responses were neuronal. Permeability studies - Colonic mucosa deprived of the muscle layer was mounted in Ussing chambers and bathed in oxygenated Krebs solution at 37°C. Functional parameters: transepithelial voltage (Vt), short-circuit current (Icc) and transepithelial resistance (Rt), were recorded in resting condition and under stimulation with CARB (100µM), forskolin and IBMX in the absence or in the presence of HT (1µM, 10µ M e 100µM). Statistical analysis was performed by paired Wilcox test and Mann-Whitney U-test (difference between groups) as appropriate. P<0.05 was considered to be statistically significant. Results: Electrically evoked contractions were linearly related to stimulation frequency in the 0.1-10 Hz. ATR and GUA inhibited electrically evoked contractions depending on stimulation frequency (p<0.05; p<0.01). The association of L-NAME, ATR and GUA increased contractions independently from stimulation frequencies (p<0.05; p<0.01).The association of HT, ATR and GUA decreased contractions at the frequency 3 -10 Hz (p<0.05). The association of HT, L-NAME, ATR and GUA decreased contractions at the frequency 1-10 Hz (p<0.05). TTX abolished electrically evoked contractions at all frequencies (p<0.05; p<0.01; p<0.001) indicating the dependence on intraluminal nerves action potentials. The exposure to HT in CARB, forskolin and IBMX stimulated tissues produced a progressive dose dependent reduction of the Vt significant at 100µM (p<0.05) and 10µM (p<0.01); a not significant reduction of the Icc at the same concentrations and a significant change in transepithelial resistance at all HT concentrations (p<0.05; p<0.01). Conclusions: HT seems to have an antioxidant effect probably through a reduction or blockage of peroxynitrite which contract the colon. Moreover it could help improve the altered intestinal permeability through a reduction of the transport activity and paracellular permeation of the human colon.