The Role And Mechanism Research Of Autophagy In The Process Of Oxidative Stress Of Lens Epithelial Cells In Age-Related Cataract

Age-related cataract is considered as one of the gerontology diseases, which can lead to severe vision impairment. Oxidative stress belongs to one of main causes of cataract. The lens epithelial cells (HLEs) suffered from ultraviolet radiation or exogenous oxidative substance produce reactive oxygen species (ROS), and give rise to lipid peroxidation and DNA damagement in tissue and cells till the emergence of cataract. Autophagy, a kind of physiological phenomena for eukaryocyte self-protection can assist cells to adapt the unfavorable stimulus and maintain the stable and self-update in cell inner environment. Up-regulation of autophagy can be achieved by activating phosphorylase AKT, which enhance migration proliferation of HLEs. Down-regulation of autophagy leads to the inhibition of above functions. Based on the reports of references, we found that there is few studies on the role of H2O2 induced reactive oxygen in regulation of autophagy for HLEs. Accordingly, this study aims to investigate the mechanism of autophagy protection to the reactive oxygen in cell line of HLEs-B3, which provides a novelty way to cataract therapy. This study includes three parts, and the main methods and results are list as below.Part 1-Screening, culture and identification of autophagy-deficient HLEs. Purpose:to obtain, culture and identify the human lens endothelial cells (HLEs) associated with autophagy deficiency. Method:Atg 7 gene was knocked out by the technique of CRISPR/Cas9 in HLEs. The steps were:(1) To confirm the target site of gene knockout; (2) To design the special DNA oligo to recognized the target site; (3) The relationship between the level of autophagy and the degree of age-related cataract. Objective:To study the correlation between the level of autophagy and the degree of age-related cataract. Methods:the levels of autophagy in the anterior capsule of lens epithelial tissue of the age-related cataract were detected by immunofluorescence technology, and the correlation between the level of autophagy and the degree of cataract was analyzed. Steps are as follows:(1) To recruit NC4 and NCI cataract cases in the experiment, according to nuclear lens opacity degree by the slit lamp evaluation; (2) To collect lens epithelial tissue specimens in the NC4 and NCI cataract cases; (3) on lens epithelial tissues, LC3 immunofluorescence labeling and detection; (4) To analyze the relationship between LC3 fluorescence intensity and degree of cataract. Results:the fluorescence intensity of LC3 was significantly enhanced in the class NC4 cataract than that in the NCI level. Conclusion:the level of autophagy is correlated with the degree of age-related cataract.Part 2-The role of autophagy in the oxidative stress induced by H2O2. Purpose: To investigate the differences of cell acitivity, oxidation, and anti-oxidation in HLEs-B3 autophagy-deficient cells and HLEs-B3 cells. Methods:PEP and A7 cells were constructed when PEP-KO and ATG7-/-plasmids were transfected into HLEs-B3 cells. Cells were divided for six group:B3 treatment, B3 control, PEP treatment, PEP control, A7 treatment, A7 control. First, for obtaining the proper level of H2O2 we compared the proliferative activity of HLEs-B3 cells after the stimulus of H2O2 at different levels. Then, we established the cell model of exogenous H2O2-related oxidative stress and focused the indexes of cell proliferation, death, oxidation, anti-oxidation, etc. (1) To study the effect of H2O2 on the cell proliferation by method of CCK-8; (2) To study the effect of H2O2 on the cell death by method of Annexin V/7-AAD; (3) To test the changes of ROS fluorescence intensity in cells by fluorescence probe of DCFH-DA; (4) To determine the differences of GSH amd MDA activity by enzymatic method. Results:(1) The CCK-8 method revealed that the level of 300μM H2O2 can inhibit the cell proliferation. The smallest decline was observed in group of B3 A7 treatment; (2) (1) The Annexin V/7-AAD method revealed that the level of 300μM H2O2 can induce the cell death. The samllest decline was observed in group of A7 treatment; (3)ROS fluorescence intensity was significant increased in group of A7 treatment by the test of flow cytometry; (4) Enzymatic method showed decrease of GSH activity in A7 group after the H2O2 stimulus, but the MDA activity was obviously enhanced. Conclusion:In the environment of oxidative stress, when the autophagy was inhibited, it would decreased anti-oxidative ability and cell death.Part 3-The mechanism reseach on the regulation of H2O2-induced oxidative stree by autophagy. Purpose:To discuss the molecular mechasim of regulation of H2O2-induced oxidative stree by autophagy. Methods:The HLEs-B3 autophagy-deficient cells and HLEs-B3 cells were treated by H2O2 for 24 h, and the indexes of Caspase-3, Caspase-8, Caspase-9、Bcl-2、Bax were tested by westen blot. Result:The data of westen blot showed that the stimulus of H2O2 can enchance the express of Caspase-3、Caspase-9、Bax in A7group, while Caspase-8 was inhibited. Conclusion:In the environment of oxidative stress, autophagy and apoptosis are in a functional reciprocal inhibition; autophagy cell death may be the main role in cell death.