Proinflammatory cytokine-induced NF-kappaB 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-kappaB (NF-kappaB) activation by proinflammatory cytokines. Silymarin exhibits anti-inflammatory and antioxidant effects but the effect of silymarin in human mesangial cells is largely unknown.


METHODS
NF-kappaB 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 flow cytometry. Monocyte chemoattractant protein-1 (MCP-1) expression was measured by Northern blot analysis and ELISA.


RESULTS
NF-kappaB was activated within 30 min by tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta). 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-1beta-induced NF-kappaB activation. Intracellular calcium was increased by TNF-alpha and IL-1beta. Furthermore, a calcium chelator, BAPTA-AM, attenuated the NF-kappaB activation. Silymarin dose-dependently inhibited the TNF-alpha- or IL-1beta-induced NF-kappaB activation and MCP-1 expression. Silymarin also inhibited TNF-alpha-induced intracellular calcium.


CONCLUSIONS
Induction of NF-kappaB within 30 min by TNF-alpha- and IL-1beta was mediated through intracellular calcium but not ROS. Silymarin inhibited TNF-alpha-induced calcium-dependent NF-kappaB activation irrespective of its antioxidant effect.
Chang
Jai Wong
JW
Department of Internal Medicine, Urology and Asan Institute for Life Sciences, College of Medicine, University of Ulsan, Seoul, Korea.
Kim
Choung Soo
CS
Kim
Soon Bae
SB
Park
Su Kil
SK
Park
Jung Sik
JS
Lee
Sang Koo
SK
D000975:Antioxidants / Q000494:pharmacology*; D015152:Blotting, Northern; D002118:Calcium / Q000502:physiology*; D018929:Cell Culture Techniques; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007150:Immunohistochemistry; D007249:Inflammation; D007375:Interleukin-1 / Q000502:physiology; D050527:Mesangial Cells / Q000187:drug effects* / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism*; D017382:Reactive Oxygen Species; D015398:Signal Transduction / Q000187:drug effects; D012838:Silymarin / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology
D000975:Antioxidants; D007375:Interleukin-1; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D012838:Silymarin; D014409:Tumor Necrosis Factor-alpha; D002118:Calcium