Reactive nitrogen species modulate the effects of rhein, an active component of senna laxatives, on human epithelium in vitro.

BACKGROUND: Senna laxatives are used worldwide. However, their misuse can lead to chronic mucosal inflammation with the accumulation of pigment-laden leukocytes and may cause colon cells to undergo apoptosis. This study explores the mechanisms by which rhein, an active component of senna, acts on a human intestinal cell line to induce ion secretion, apoptosis, and indirect chemotaxis of polymorphonuclear leukocytes. METHODS: Human colonic adenocarcinoma (CaCo-2) monolayer cells, in the presence or in the absence of rhein, were used to monitor the production of reactive nitrogen species using the Griess reaction. Modified Ussing chambers were used to study electrolyte secretion. The capacity to recruit human polymorphonuclear leukocytes was evaluated using masked well chemotaxis chambers. Rhein-induced apoptosis was investigated by counting apoptotic nuclei stained with Hoechst 33258 dye. RESULTS: Rhein caused a dose-dependent increase in short-circuit current that was abolished in chloride-free bathing buffer or by preincubating with 100 micromol/L NG-nitro-L-arginine (L-NAME) methyl ester. The concentration that maximally stimulated intestinal secretion, 50 micromol/L rhein, induced nitrate production. Supernatants obtained from CaCo-2 cultures after incubation with 50 micromol/L rhein stimulated a time-dependent polymorphonuclear leukocytes chemotaxis that was significantly decreased with 100 micromol/L L-NAME, whereas rhein per se was not active. Neutralizing antibodies anti-interleukin-8 (IL-8) and anti-ENA78 also inhibited chemotaxis. Overnight rhein incubation produced an increased number of apoptotic cells in the culture supernatant that was significantly decreased by preincubation with 100 micromol/L L-NAME. Light-degraded rhein had no effects on CaCo-2 monolayers. CONCLUSIONS: The integrity of rhein is crucial to generating nitric oxide, which mediates, with different time courses, ion secretion, chemotaxis, and apoptosis of human-derived cells.

Unconjugated bile acids modulate adult and neonatal neutrophil chemotaxis induced in vitro by N-formyl-met-leu-phe-peptide.

In this study, we have investigated the effect of hydrophobic and hydrophilic unconjugated bile acids (UBAs)-ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), lithocholic acid, and colic acid-on chemotaxis in adult and neonatal human polymorphonuclear leukocytes (PMNs). The trypan blue exclusion dye test was preliminarily performed to determine the toxicity of the studied UBAs on PMNs. N-formyl-methionyl-leucyl-phenylalanine (100 nM) was used as a chemoattractant. Chemotaxis (1 x 10(6)cells/mL) was analyzed in the presence or absence of UBAs (10 microM) by blind well chambers. The antioxidants vitamin E and vitamin C were tested for their ability to reduce the inhibitory effect of UBAs. We found that only CDCA was able to induce damage in PMNs in the range of 1-40 microM. Both CDCA and UDCA were able to inhibit chemotaxis in PMNs, whereas lithocholic acid and colic acid were ineffective. The inhibitory effect was reversible inasmuch as PMNs incubated with either CDCA or UDCA and subsequently washed showed normal chemotaxis. Concomitant incubation of PMNs with UBAs and vitamins C or E reversed the inhibition. We did not find substantial differences between PMNs from adults or newborns. In conclusion, CDCA and UDCA are able to reduce, in a specific and reversible fashion, both adult and newborn neutrophil chemotaxis. As concomitant incubation of UBAs and electron scavengers restores PMN chemotaxis to control values, we conclude that free radicals may be involved in the mechanism of inhibition. We speculate that this defect may contribute to the impaired host response described in cholestatic patient.