Protective Effect Of Rutin Against H₂O₂-induced Oxidative Stress And Apoptosis In Human Lens Epithelial Cells And The Possible Mechanisms

Purpose Cataract, as the major etiology for decrease of vision and eventually blindness in the elderly population, has become a world-wide problem in public health which cannot be ignored. The apoptosis of lens epithelial cell is the common cellular basis for the occurrence and progress of non-congenital cataract in both animal and human being, and reactive oxygen species are major contributors to the apoptosis of lens epithelial cells. Thus, the inhibition of oxidative stress and apoptosis of epithelial cells plays an important role in the prevention of cataract formation. Based on the fact that rutin has been shown to have significant property of eliminating many different kinds of free radicals, in our studies, we will establish the oxidative stress model in human lens epithelial cell to systemically research the protective effect of natural anti-oxidant rutin against H₂O₂-induced oxidative stress and apoptosis, and study the mechanism involved by using different index system for oxidative stress and apoptosis. Our study will provide a new research direction for clinical and pharmacological treatment in the early cataract formation and prognosis.Methods1. SRA01/04 HLECs in the logarithmic growth phase were collected in experiments when they had grown to 80%- 90% confluence,then the cells were treated with the different concentrations（0,50,100,200,400,800μM） of H₂O₂ or different concentrations of rutin（0,25,50,100,200,400μM） for 24 h, cell viability was assessed by MTT assay. And we choose the appropriate concentrations of H₂O₂ and rutin according the above experiment results.2. Cells were exposed to H₂O₂ for 24 h without or with rutin pretreatment at different concentrations（25,50,100μM）. Cell viability was assessed by MTT assay,morphological changes were examined by inverted microscope,cell migration rate was measured by scarification test.3. To evaluate the protective effect of rutin against oxidative-stress, the level of reactive oxygen species（ROS） was examined using DCFH-DA 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 ELISA.4. To evaluate the anti-apoptosis ability of rutin at the morphological and the molecular level, cell apoptosis was determined by flow cytometry, DAPI staining assay and DNA fragmentation assay.5. To determine whether rutin plays a role through regulating mitochondria-dependent Bcl-2/Bax famlily, the loss of mitochondria membrane potential（ΔΨm）were detected by flow cytometric analyses after JC-1 staining.The expression change of Bcl-2, Bax and caspase-3 at protein levels were detected by western blot analysis respectively.6. To determine whether NF-κB signaling is involved in the process of apoptosis of lens epithelial cells induced by H₂O₂, cells were exposed to H₂O₂ for 24 h without or with100μM rutin/100μM PDTC. Activation and translocation of nuclear factor kappa-B（NF-кB /p65） were examined by immunocytochemistry. The expression change of nuclear NF-кB /p65 and total IκB at protein levels were detected by Western blot analysis respectively.7. To determine whether MAPK signaling is involved in the process of apoptosis of lens epithelial cells induced by H₂O₂, cells were exposed to H₂O₂ for 24 h without or with 100μM rutin/ 20μM PD98059/ 10μM SP600125/10μM SB203580. The expression change of p-ERK, p-p38, p-JNK at protein levels were detected by western blot analysis respectively.Results1. In H₂O₂-treated cells the cell vialibity and migration rate were lower significantly than those of the control cells. Compared with H₂O₂-treated group, pretreatment with rutin at various concentrations significantly increased cell vialibity and migration rate（P<0.05）The effects were positively associated with the increased concentration of rutin. In rutin pretreated cells, the number of cells with morphological abnormality was lower obviously than that of H₂O₂-treated group. These suggested that rutin may inhibit the H₂O₂-induced injury of cell, enhance the cell viability and stablize the cell morphology.2. In the presence of rutin, H₂O₂-induced intracellular excessive ROS and MDA were attenuated, whereas intracellular SOD and GSH depletion were prevented. Rutin surpressed H₂O₂-induced generation of intracellular ROS and lipid peroxidation and protected the antioxidant system.3.Compared with H₂O₂-treated cells, pretreatment with rutin at various concentrations prevented the apoptosis induction in a dose-dependent manner（P<0.01）by flow cytometry analysis. DNA fragmentation assay showed that DNA extracted from the H₂O₂-treated HLE cells displayed the typical DNA laddering pattern, while DNA from the control cells and rutin-pretreated cells displayed very little or no DNA fragmentation pattern. DAPI staining assay showed in H₂O₂-treated group, the numbers of shrunken, irregular and fragmented nuclei were increased obviously as compared to the control cells and the changes were inhibited by pretrement with rutin.These suggested that rutin protected HLECs against apoptosis not only at the morphological level but also at the molecular level.4. Mitochondrial membrane potential（ΔΨm） of the cells pretreated with rutin exhibited an obvious increase compared to H₂O₂-treated cells.（P<0.05）, the effects were positively associated with the increased concentration of rutin. In H₂O₂-treated cells the protein levels of caspase-3 and Bax were greater and the level of Bcl-2 was lower than those of the control cells.（P<0.01）. Compared with H₂O₂-treated group, pretreatment with rutin at various concentrations significantly inhibited caspase-3 and Bax protein expression, but promoted Bcl-2 expression（ P<0.05）.These results suggest that rutin can stablized the H₂O₂-induced loss of ΔΨm, surpress H₂O₂-induced apaptosis through the mitochondria-dependent Bcl-2/Bax family5. Rutin blocked the activation and translocation of NF-кB /p65 induced by H₂O₂. Western blot analysis indicated that exposure to H₂O₂ resulted in an increase of nuclear NF-κB/p65 protein and a decrease of total IκB as compared to control cells, and the effect was inhibited by the pretreatment with rutin or PDTC.（ P<0.01） Immunocytochemistry assay clearly showed the translocation of NF-κB/p65 from cytoplasm to nucleus, and the translocation was inhibited by pretreatment with rutin or PDTC. This implies that rutin may offer protection against oxidative stress-induced apoptosis by regulating NF-κB singal pathways.6. Western blot analysis indicated that exposure to H₂O₂ resulted in an increase of phosphorylation of ERK, p38, JNK protein as compared to control cells（ P<0.01）, and the effect was inhibited by the pretreatment with rutin /PD98059/SB203580/ SP600125.（ P<0.01）.This implies that rutin may offer protection against oxidative stress-induced apoptosis by regulating MAPKs singal pathways.ConclusionsRutin inhibits H₂O₂ induced oxidative stress of lens epithelial cells by decreasing the ROS, reducing lipid peroxidation, and protecting the intracellular antioxidant system, while rutin inhibits the H₂O₂-induced apoptosis of lens epithelial cells by stabilizing the mitochondrial membrane potential（ΔΨm）, up-regulating Bcl-2 expression, while down-regulationg Bax, caspase-3 expression. NF-κB and MAPK signaling pathway is involved in the process of apoptosis of lens epithelial cells induced by H₂O₂...