Purpose: The aim of this study was to evaluate the effect of rutin on oxidative stress and apoptosis induced by H2O2 in human lens epithelial (HLE) cells and the associated mechanisms involved.
Methods: Cell viability was assessed by 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and cell apoptosis was determined by flow cytometry, TUNEL assay and DNA fragmentation assay after 24 h treatment of 100 μM H2O2 with or without rutin pretreatment at various concentrations. The level of reactive oxygen species (ROS) was examined using 2',7'-dichlorodihydrofluorescein diacetate by flow cytometry. The activity of superoxide dismutase (SOD) was measured by xanthinoxidase method and the contents of glutathione (GSH) and malondialdehyde (MDA) were quantified by enzyme-linked immunosorbent assay. The expression change of Bcl-2, Bax and caspase-3 at mRNA and protein levels were detected by real-time polymerized chain reaction (RT-PCR) and Western-blot analysis, respectively. Activation and translocation of nuclear factor-kappaB (NF-кB/p65) were examined by Western blot and immunocytochemistry.
Results: Rutin pretreatment protected HLE cells from H2O2-induced cell viability decrease and apoptosis. In addition, in the presence of rutin, H2O2-induced intracellular excessive ROS and MDA were attenuated, whereas intracellular SOD and GSH depletion were prevented. Moreover, rutin also inhibited the up-regulation of caspase-3 and Bax expression and rescued down-regulation of Bcl-2 expression. Lastly, rutin blocked the activation and translocation of NF-кB/p65 induced by H2O2.
Conclusions: Our results demonstrated that rutin effectively protects HLE cells from H2O2-induced oxidative stress and apoptosis in a dose-dependent manner. The involved mechanisms may be related to the regulation of ROS production, the inhabitation of lipid peroxidation, the protection of intracellular antioxidant system and its modulation of Bcl-2/Bax family and NF-кB/p65 signaling pathway.
Keywords: Antioxidant; apoptosis; cataract; human lens epithelial cells; rutin.