The Effect Of Chinese Phytoestrogens On Human Lens Epithelial Cell Damaged By Oxidation

ObjectiveTo investigate the effect of Chinese medicine with estrogen activity,isopsoralen(ISR) and ecdysterone(ECR),on human lens epithelial cell damaged by H₂O₂ oxidation.To reveal the cellular and molecular mechanism of preventing and treating cataract by phytoestrogen in the fields of oxidation and apoptosis,in order to provide scientific evidence for pursue effective midicine to prevent and treat cataract.MethodHuman lens epithelia cell B₃(HLE-B₃) were cultured with H₂O₂ to duplicate the model of oxidative damage,meanwhile,HLE-B₃ were cultured with ISR,ECR and E₂ respectively.Then,the damaged HLE-B₃ were used for following studies:1.The effect of different concentrations of ISR and ECR on activity of HLE-B₃ damaged by H₂O₂ were detected by methyl thiazolyl tetrazolium(MTT).2.The activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-px),catalase (CAT) and the content of Malonaldehyde(MDA) of HLE-B₃ damaged by H₂O₂ were measured via chemical chromatometry to study the anti-oxidative effects of ISR and ECR and the enzymatic mechanism.3.The effect of ISR and ECR on morphology of HLE-B₃ damaged by H₂O₂ was observed under transmission electron-microscope.4.The effect of ISR and ECR on nucleus DNA content of HLE-B₃ damaged by H₂O₂ was analyzed using flow cytometry to study the nulear mechanism of apoptosis.5.The effect of ISR and ECR on mitochondrial transmembrane potential(△ψM) of HLE-B₃ damaged by H₂O₂ was analyzed using flow cytometry to study the cytoplasm mechan ism of apoptosis.Results1.The result of MTT test show that the optical density values(OD) of H₂O₂ group was lower obviously than that in control group(p<0.01).The OD values of HLE-B₃ in different group of ISR and ECR were increased obviously to compare with H₂O₂ group,The results indicated dosedependence.2.The result of chemical test show that the activities of SOD,GSH-px and CAT decreasedin H₂O₂ group,however,the activities of SOD,GSH-px and CAT increased markedly in E₂,ISR and ECR group compared with H₂O₂ group(p<0.01).The MDA contents increased in H₂O₂ group,while it decreased markedly in E₂,ISR and ECR group compared with H₂O₂ group(p<0.01).3.The morphological changes of apoptosis by transmission electron microscopy showed that the typical morphological changes of apoptosis were observed under transmission electronmicmscope,such as heterochromatin condensation and aggregated at the nuclear periphery,and nuclear fragmentation as well in HLE-B₃ induced by H₂O₂.However,E₂,ISR and ECR group can alleviated these changes of morphology.4.The result of flow cytometry showed that DNA content decreased in H₂O₂ group comparing with control group,and DNA content increased and the apoptosis rate decreased markedly in E₂,ISR and ECR group comparing with H₂O₂ group(p<0.01).5.The result of flow cytometry showed that mitoehondrial transmembrane potential(△ψM) in H₂O₂ group reduced obviously compared to control group(p<0.01)and△ψM in E₂,ISR and ECR group is increased remarkably compared to H₂O₂ group(p<0.01).Conclusion1.H₂O₂ Can induce oxidative damages of HLE-B₃.2.ISR and ECR can safeguard oxidative damages,the increases of SOD,GSH-px,CAT activity and decrease of MDA content is the important mechanism.3.H₂O₂ can induce apoptosis of HLE-B₃.ISR and ECR can reduced apoptosis of HLE-B₃ in duced by H₂O₂.Increase of nucleus DNA content and decrease of apoptosis rate may be one of the important mechanism.4.Increase of mitochondrial transmembrane potential(△ψM) through cytoplasm pathway may be the another important mechanism of decreasing apoptosis by ISR and ECR.