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) mono-layer 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 μmol/L N^G-nitro-L-arginine (L-NAME) methyl ester. The concentration that maximally stimulated intestinal secretion, 50 μmol/L rhein, induced nitrate production. Supernatants obtained from CaCo-2 cultures after incubation with 50 μmol/L rhein stimulated a time-dependent polymorphonuclear leukocytes chemotaxis that was significantly decreased with 100 μmol/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 μmol/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.