The Protective Effect Of Grx2 In Blue Lightinduced Retinal Damage And Mechanism Research

Objectives:1.We aim to establish blue light-induced mouse retinal damage model and detect the expression of Grx2 in retinal tissues.2.We aim to investigate the effect of Grx2 on the photodamage of retina induced by blue light in mice.3.We aim to establish a model of light injury of human retinal pigment epithelial cells induced by blue light,and investigate the role and mechanism of Grx2 in the damage of ARPE-19 cells induced by blue light.Methods:1.The C57BL/6J mice were illuminated using a blue light device with an intensity of 2000 Lux for continuous light 3 days and 5 hours a day to establish a mouse retinal light injury model.The function of mouse retina was detected by ERG,the structure of mouse retina was observed by HE staining,and the m RNA and protein expression levels of Grx2 in mouse retina were detected by real-time PCR and Western blotting.2.Grx2 knockout and knock-in mice were obtained by CRISPR/Cas9 technology,the genotype of transgenic mice was identified by agarose gel electrophoresis,and the expression of Grx2 protein in the retina of transgenic mice was verified by Western blotting.ERG was used to detect mouse retinal function,HE staining was used to observe mouse retinal tissue structure,and Western blotting was used to detect mouse retinal apoptosis-related factors and JNK signaling pathway protein levels.3.The ARPE-19 cells were illuminated 0 h,6 h,12 h,24 h and 48 h with 1500 Lux of blue light.CCK-8 was used to detect the cell viability of ARPE-19 cells induced by blue light under different light time points,the LDH release test was used to detect cytotoxicity,oxygen detection kit was used to detect total intracellular ROS,mito SOX? was used to detect mitochondrial ROS.Real-time PCR was used to detect Grx2 m RNA expression levels in ARPE-19 cells after blue light,and Western blotting was used to detect Grx2 and apoptosis-related protein expressions after blue light.4.ARPE-19 cells were infected with lentivirus to establish Grx2 overexpression and low expression models,which were divided into CN group,CN+BL group,si Grx2+BL group,si NC+BL group,oe Grx2+BL group and oe NC+BL group.The blue light irradiation time was 24 h.CCK-8 was used to detect the cell viability of ARPE-19 cells induced by blue light,the LDH release test was used to detect cytotoxicity,the apoptosis detection method(Annexin-V FITC/Propidium Iodide)was used to detect the apoptotic rate,and mito SOX? was used to detect mitochondrial ROS.Normal ARPE-19 cells and lentivirus infected cells were divided into CN+BL group,si Grx2+BL group,si Grx2+SP+BL group,oe Grx2+BL group and oe Grx2+Ani+BL group.Western blotting was used to detect the expression levels of apoptosis-related proteins and the protein levels of JNK signaling pathway after light exposure.Results:1.In our results,after irradiating C57BL/6J mice with 2000 Lux blue light,the ERG results showed that the a wave and b wave amplitude of dark adaptation and the b wave amplitude of light adaptation were significantly reduced,and the mouse retinal function was significantly impaired.HE results showed that the retinal tissue structure was damaged and manifested as obvious thinning of outer nuclear layer.Grx2 m RNA and protein levels in mouse retinal tissue were increased significantly.2.Agarose gel electrophoresis identified the genotypes of Grx2 knockout and knock-in mice successfully.The ERG results showed that the amplitude of a wave,b wave in the dark adaptation and b wave in the light adaptation of Grx2 knockout mice were significantly reduced after light,while the amplitude of a wave,b wave in the dark adaptation and b wave in the light adaptation of Grx2 gene knock-in mice had no significant reduction.HE results showed that blue light-induced significant thinning of the outer nuclear layer of the retina in mice after Grx2 knockout,while there was no significant change in the thickness of the outer nuclear layer of the retina induced by blue light after Grx2 knock-in.Western blotting results showed that Bax,Cleaved Caspase 3,Cleaved Caspase 9,Cytochrome c,and p-JNK protein levels increased significantly after light exposure,and Bcl-2 protein decreased significantly.After Grx2 was knocked out,the corresponding protein changed more obviously.While Grx2 knocked-in,the protein levels of Bax,Cleaved Caspase 3,Cleaved Caspase 9,Cytochrome c and p-JNK were significantly reduced,and the Bcl-2 protein level was significantly increased.3.After blue light irradiation of ARPE-19 cells,with the prolonged blue light irradiation time,the cell viability of ARPE-19 cells decreased,and cytotoxicity and apoptosis increased significantly.In addition,blue light could stimulate a significant increasement in total ROS and Mt ROS in cells,and aggravate oxidative stress in a certain time-dependent manner.Grx2 m RNA and protein expressions increased significantly after blue light irradiation.The expression of Grx2 m RNA peaked at 12 h under blue light,and the level of Grx2 protein expression at 24 h under blue light was higher than that at 12 h.The protein expression of Bax,Cleaved Caspase 3 and Cleaved Caspase 9 increased in a time-dependent manner after blue light illumination,while Bcl-2 decreased in a time-dependent manner.4.Grx2 overexpression can alleviate the oxidative stress response in ARPE-19 cells induced by blue light,improve cell viability,reduce LDH release,reduce cytotoxicity,increase mitochondrial membrane potential,and reduce cell apoptosis rate.Moreover,compared with CN+BL group,the p-JNK protein level in CN+SP+BL group decreased significantly,while the p-JNK protein level in CN+Ani+BL group increased significantly.The p-JNK level in si Grx2+SP+BL group was significantly lower than that in si Grx2+BL group.The p-JNK level in oe Grx2+Ani+BL group was significantly higher than that in oe Grx2+BL group.In addition,the Bax,Cleaved Caspase 3,Cleaved Caspase 9 and Cytochrome c protein levels in si Grx2+SP+BL group were significantly lower than those in si Grx2+BL group,while the protein expression of Bcl-2 was increased significantly.The protein levels of Bax,Cleaved Caspase 3,Cleaved Caspase 9 and Cytochrome c in oe Grx2+Ani+BL group were significantly higher than those in oe Grx2+Ani+BL group,while the protein expression of Bcl-2 was decreased significantly.Conclusion:1.Blue light could damage the structure and function of the mouse retina significantly,and increase the expression of Grx2 in the mouse retina.2.Grx2 knockout will aggravate blue light-induced damage to mouse retinal structure and function,activate JNK signaling pathway,and promote cell apoptosis,while Grx2 knock-in will reduce blue light-induced damage to mouse retina,down-regulate JNK signaling pathway and inhibit apoptosis.3.Blue light could cause ARPE-19 cell damage,and this damage is time-dependent.The longer blue light illumination time,the more severe cytotoxicity,the lower cell viability,and the increase of total intracellular ROS and mitochondrial ROS,which in turn causes apoptosis.4.Low expression of Grx2 reduced the cell viability of blue-induced ARPE-19 cells and aggravated cell apoptosis significantly,while overexpression of Grx2 could improve the damage of blue light-induced ARPE-19 cells,increase cell viability and inhibit cell apoptosis.Grx2 plays a protective role in blue light-induced ARPE-19 cell oxidative damage,and this protective effect may be related to the inhibition of activation of JNK signaling pathway.