Proinflammatory cytokine-induced NF-κB activation in human mesangial cells is mediated through intracellular calcium but not ROS:: Effects of silymarin

Background: It is not fully understood whether intracellular calcium and/or reactive oxygen species (ROS) are involved in nuclear factor-kappa B (NF-kappa B) activation by proinfl ammatory cytokines. Silymarin exhibits antiinflammatory and antioxidant effects but the effect of silymarin in human mesangial cells is largely unknown. Method: NF-kappa B binding activity was measured by electrophoretic mobility shift assay. Intracellular calcium was monitored by confocal microscopy using Fluo-3 and intracellular ROS production was determined by fl ow cytometry. Monocyte chemoattractant protein-1 (MCP-1) expression was measured by Northern blot analysis and ELISA. Results: NF-kappa B was activated within 30 min by tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). Intracellular ROS was not produced until 30 min and also antioxidants such as N- acetylcysteine and tiron had no effect on the TNF-alpha- or IL-1 beta- induced NF-kappa B activation. Intracellular calcium was increased by TNF-alpha and IL-1 beta. Furthermore, a calcium chelator, BAPTA-AM, attenuated the NF-kappa B activation. Silymarin dose-dependently inhibited the TNF-alpha- or IL-1 beta- induced NF-kappa B activation and MCP-1 expression. Silymarin also inhibited TNF-alpha-induced intracellular calcium. Conclusions: Induction of NF-kappa B within 30 min by TNF-alpha- and IL-1 beta was mediated through intracellular calcium but not ROS. Silymarin inhibited TNF-alpha-induced calcium-dependent NF-kappa B activation irrespective of its antioxidant effect. Copyright (c) 2006 S. Karger AG, Basel.