ABSTRACT
Objective:To explore the role of Grx2 in the pathogenesis of cataract by establishing Grx2 knockout (KO) and knockin (KI) mouse models. Methods:Ten black C57BL/6J mice were selected to make Grx2 KO model ( n=5) and Grx2 KI model ( n=5) using CRISPR/Cas9 system.The offspring mice were sequenced by tail clipping and included in the corresponding experimental group according to the genotype.The general condition and lens opacity was recorded.After the mice were sacrificed, the pathological changes of lens were observed by hematoxylin-eosin staining.The contents of reactive oxygen species (ROS) and 8-hydroxy-desoxyguanosine (8-OHdG) were analyzed by enzyme-linked immunosorbent assay (ELISA).The relative expression levels of Grx2, glutathione (GSH), B-cell lymphoma-2 (Bcl-2) , glutathione disulfide (GSSG) and Bcl-2-associated X protein (Bax) in mice lens were assayed.The use and feeding of experimental animals were in accordance with the Regulations on the Management of Experimental Animals issued by the State Science and Technology Commission.The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (No.2020-125). Results:The offspring of Grx2 KO and Grx2 KI homozygous and heterozygous mice were confirmed by tail cutting nested PCR and gene sequencing.Compared with the wild type (WT) mice of same age, the lens opacity of Grx2 KO heterozygous mice occurred earlier, while the lens of Grx2 KI homozygous mice remained transparent all the time.A large number of gaps and vacuoles were found in the lens fibers of 5-month-old Grx2 KO mice.The 8-OHdG content and ROS fluorescence intensity in the lens of 5-month-old Grx2 KO mice were (3.886±0.326)ng/ml and 1 594±132, which were significantly higher than (3.531±0.250)ng/ml and 1 157±123 in WT mice ( t=2.711, P=0.033; t=3.384, P=0.028).The relative expression levels of Grx2, GSH and Bcl-2 in the lens of 5-month-old Grx2 KO mice were 0.23±0.01, 0.70±0.06 and 0.32±0.03, which were significantly lower than 0.52±0.02, 1.04±0.08 and 0.49±0.04 of WT mice ( t=2.815, P=0.020; t=2.457, P=0.033; t=2.279, P=0.041). Conclusions:Grx2 KO and Grx2 KI mouse models are successfully established in this study.The occurrence and development of age-related cataract are accelerated in Grx2 KO mice.
ABSTRACT
Objective To observe the influences of hyperoxia on the migration of human lens epithelial cells (HLECs) and the inhibitory effects of glutathione 2 (Grx2) on excessive migration of HLECs induced by hyperoxia.Methods The HLECs were divided into the hyperoxic model group and normoxic control group.HLECs were cultured in 80％ oxygen mixture for 2 days to establish the oxidative damage model of HLECs induced by hyperoxia in vitro (hyperoxia model group),and cultured in normoxia mixture as the normoxic control group.Cell migration was observed by scratch test,cytoskeleton morphology was observed by phalloidin staining,and Grx2 mRNA level was detected by real-time quantitative PCR.HLECs in the hyperoxic model group and the normoxic control group were subdivided into empty plasmid transfection group and Grx2 plasmid transfection group,respectively.The migration behavior and cytoskeleton changes of hyperoxic model cells in different transfection groups were observed.MitoSOX staining was used to detect reactive oxygen species (ROS) content of mitochondria under flow cytometry,and JC-1 staining was used to observe the changes of mitochondrial membrane potential under laser-scanning confocal microscopy.Results In the process of cell culture,the cells grew well.Twenty-four hours and 48 hours after scratching,the scratch distance of the hyperoxic model group was smaller than that of the normoxic control group,with significant differences between them (both at P＜0.05).The cell circumference of the hyperoxic model group was increased compared with that of the normoxic group,with a significant difference between them (t =-2.254,P＜0.05).After 2 days culture,the relative expression level of Grx2 mRNA in the hyperoxic model group was 0.54±0.11,which was significantly lower than that in the normoxic control group (t =7.128,P＜0.01).The transfection efficiency of Grx2 was higher than 90％.At 24 hours and 48 hours after scratching,the scratch distance in the Grx2 transfected hyperoxic model group was wider than that in the empty plasmid transfected hyperoxic model group,with significant differences between them (both at P ＜ 0.05).The relative expression level of Grx2 mRNA in Grx2 transfected cells was up-regulated compared with that in empty plasmid transfected cells,with a significant difference between them (P＜0.01).Compared with the normoxic control group,the cell circumference of the hyperoxic model group was decreased,with a significant difference between them (P＜0.05).The relative mean fluorescence intensity of mitochondrial ROS in the Grx2 transfected hyperoxic model group was lower than that in the empty plasmid transfected hyperoxic model group,with a significant difference between them (P＜0.05).Under the hyperoxic condition,the red/green fluorescence intensity of Grx2 transfected group was higher than that of the empty plasmid transfected group,with a significant difference between them (P＜0.01).Conclusions Grx2 may inhibit the excessive migration of HLECs under hyperoxia by regulating oxidative stress in mitochondria.