Rutoside Protects Human Lens Epithelial Cells Against Oxidative Injury And Apoptosis Via Regulating Related Genes Expression

Objective:Domestic and foreign scholars have considered that oxidative damage is thecommon mechanism of age-related cataract in various risk factors. And lens epithelialcell （LEC） apoptosis appears to be a common cellular basis for non-congenital cataractdevelopment in humans and animals. The expression of Bcl-2is negative while the Baxand Caspase-3were positive in age-related cataract lens epithelial cell. Based on theabove theory, our current study was designed to investigate the protective effect offlavonoid compound rutoside （Ru） on hydrogen peroxide-induced oxidative injury andapoptosis, and the expression of apoptotic gene Bcl-2, Bax, Caspase-3on transcriptionallevel in human lens epithelial cells （HLEC）.Methods:（1） Normal SRA01-04line HLEC was treated with different concentration of Ru andH₂O₂for24h. The vitality of the cells was then detected by MTT to determine theeffective experimental concentration of Ru and H₂O₂.（2） Normal HLEC was pretreated with different concentration of Ru（0,25,50,100μmol/L）for1h, followed by treatment with200μmol/L H₂O₂for24h. Theproliferative activity of each group cells was then detected by MTT.（3） The activities of superoxide dismutase （SOD） in each group culture medium weredetected with colorimetric methods, and the content of glutathione （GSH） andMalondialdehyde （MDA） were measured by ELISA.（4） The apoptotic cells were detected by TUNEL stain. The cells morphology wereobserved by the optical microscopy and then calculating the apoptosis index （AI）. （5） Extracting the total RNA in the cell supernatant and detecting the expression ofBcl-2, Bax, Caspase-3mRNA by Real-Time Quantitative PCR.Results:（1） It is found that the cell inhibition rate was close to the half inhibition rate （IC50）when the H₂O₂concentration was at200μmol/L. Therefore200μmol/L H₂O₂was usedin the following experiments.（2） Ru, at concentration of0¹00umol/l, has no significant influence to the cell vitality.Therefore, we chose25,50,100μmol/L as the pretreated concentration in the Ru lowdose group, middle dose group and the high group.（3） The proliferation ability of HLEC and the content of SOD, GSH in H₂O₂group weresignificantly lower than that in control group, while MDA and AI increased evidently（Pvalue＜0.01）; The expression level of Bcl-2mRNA in H₂O₂group was significantlylowered while Bax and Caspase-3mRNA were increased（P value＜0.05）.（4） Ru （25,50,100μmol/L） pretreated group being compared with H₂O₂group，its cellviability and antioxidant levels raised obviously, and the apoptosis cell quantitydiminished significantly（P value＜0.01）. Correspondingly, the expression level ofBcl-2mRNA was up-regulated, and the Bax and Caspase-3mRNA level weredown-regulated（P value＜0.05）.ConclusionRu can improve the cell proliferation ability and protects hydrogenperoxide-induced oxidative injury and apoptosis in human lens epithelial cells. Themechanism might be associated with its up-regulating the expression level of Bcl-2mRNA and down-regulating the transcriptional level expression level of Bax,Caspase-3.