Research Of Chk2 On The Protection For Retinal Pigment Epithelium Under Oxidative Stress Through Promoting Autophagy

Objective:Age-related macular degeneration（AMD）is a blinding disease caused by multiple factors and it is the main cause of vision loss in the elderly.Dry AMD is one of the subtypes.Its pathogenesis has not been fully clarified,and there is still a lack of effective prevention and treatment methods for it clinically.Cell cycle checkpoint kinase2（Chk2）is an evolutionarily highly conserved serine/threonine protein kinase that plays an important role in maintaining cellular homeostasis,but its mechanism of action in dry AMD is still unclear.Therefore,this study explored the mechanism of action of Chk2 in dry AMD.Methods:1.The microarray data GSE29801 of AMD-related gene expression profile was downloaded from the National Center of Biotechnology Information（NCBI）GEO database.61 samples from the Dry AMD group and 151 samples from the normal group were selected as the total research samples.Then,we standardized the gene expression matrix,obtained the normalized expression profile data of all chip samples,screened and obtained differentially expressed gene sets,and performed differential gene ontology and signal pathway significance enrichment analysis.2.Vitro experiments were conducted by using Chk2 siRNA to interfere with human retinal pigment epithelial cells（ARPE-19）which were stimulated with H₂O₂ to simulate the dry AMD oxidative stress cell model.Then,we detected ROS level and apoptosis rate of the cells by flow cytometry.And,the expression of Chk2,phosphorylated Chk2（p-Chk2）,P62,LC3Ⅰ,LC3Ⅱin cells was analyzed by Western blot.3.Chk2 heterozygous mice(Chk2^+/-)were breeding,hybridized to obtain Chk2 knockout（Conventional Knockout,KO）mice(Chk2^-/-)and WT mice(Chk2^-/-).Group settings were as followed:WT group,Chk2-KO group,WT+NaIO₃ group,Chk2-KO+NaIO₃ group,5 mice in each group.We intraperitoneally injected sodium iodate（35mg/kg）into mice to simulate dry AMD in vivo model.And,5 days later,the eyeballs were taken and stained by HE staining.The retinal histopathological morphology of each group was detected,and the expression of P62 was analyzed by immunofluorescence staining in the same group setting.Results:1.Through bioinformatics analysis,the results of differentially expressed gene analysis showed that compared with the control samples,there were 1443 DEGs in the Dry AMD group（the difference was greater than 1.2 times and P was less than 0.05）,of which708 were up-regulated and 735 were down-regulated.Among them,Chk2 was significantly down-regulated in Dry AMD group compared with control samples,with P=0.038 and log FC（fold change）=-0.469.Gene Ontology（Go）enrichment analysis showed that in terms of cellular components（CC）,genes were significantly enriched in cellular components such as endoplasmic reticulum cavity,endocytic vesicle,and cytoplasmic vesicle cavity（P adjust<0.05）;In terms of biological processes（BP）,genes were significantly enriched in biological processes such as cellular oxidative detoxification,hydrogen peroxide metabolism,and response to toxic substances（P adjust<0.05）;In terms of molecular functions（MF）,genes were significantly enriched in molecular functions such as cytokine binding,antioxidant activity,peroxidase activity,and cargo receptor activity（P adjust<0.05）.KEGG signaling pathway enrichment analysis significantly enriched PI3K-AKT signaling pathway,MAPK signaling pathway and TGF-βsignaling pathway,etc.（P<0.05）.2.The results of Western blot in vitro cell experiments showed that with the increase of H₂O₂,the protein of p-Chk2 was gradually increased,the protein of P62 decreased,and the ratio of LC3Ⅱ/LC3Ⅰincreased（P<0.01）.Compared with control group,knockdown of Chk2 in ARPE-19 cells can significantly increase the level of ROS induced by H₂O₂-induced oxidative stress（P<0.01）,and the knockdown of Chk2 can promote the apoptosis of ARPE-19 cells,too（P<0.01）.The analysis of Western blot showed that,compared with the scramble+H₂O₂ group,the expression of P62 in the si Chk2+H₂O₂ group was increased,and the ratio of LC3Ⅱ/LC3Ⅰwas decreased（P<0.01）.3.The results of HE staining in Chk2 knockout mice showed that compared with WT group,there was no significant difference in retinal tissue morphology in Chk2-KO group.The mouse retina showed well-organized and clear retinal layer boundaries,in which the arrangement is regular and uniform,the density is uniform,the retinal pigment epithelium is smooth,and the pigmentation is uniform.After NaIO₃ treatment,the normal structure of RPE layer disappeared,and the boundary of retinal layer was irregular.Compared with the WT+NaIO₃ group,the abnormal black deposits were observed to increase in the retinal pigment epithelium in the Chk2-KO+NaIO₃ group.The results of immunofluorescence staining showed that compared with WT group,P62 increased after NaIO₃ treatment;compared with WT+NaIO₃ group,P62 increased significantly in Chk2-KO+NaIO₃ group（P<0.01）.Conclusion:The results of bioinformatics analysis showes that the expression of Chk2 is down-regulated in the samples of dry AMD patients.Chk2 protects retinal pigment epithelial cells under oxidative stress by promoting autophagy.Chk2 is involved in the process of retinal oxidative stress injury in mice,and its knockout inhibits autophagy and exacerbates accumulation of abnormal deposits in the retinal pigment epithelial layer in mice.