Protective Effects And Mechanism Of Nicotinamide Riboside In H₂O₂-Induced Lens Oxidative Damage

Objective:To explore the anti-oxidative effect and related protective mechanism on protecting lens from oxidative stress by nicotinamide riboside（NR）,we established a hydrogen peroxide（H₂O₂）-induced oxidative damage model of human lens epithelial cell line（SRA01/04）in vitro.Methods:In this study,cells were divided into three groups:control group（CON）,model group（H₂O₂）and treatment group（NR+H₂O₂）.After SRA01/04 cells were treated with drugs,MTT test and Hoechst staining were used to detect cell proliferation level,Annexin V-FITC/PI staining was combined with flow cytometry to detect cell apoptosis,and DCFH-DA probe was used to detect reactive oxygen species（ROS）level,scratch test to detect cell migration ability.Western blot was used to detect the expression of apoptosis and related signaling pathway proteins,while mitochondria were stained by Mito-Tracker to detect mitochondrial membrane potential（MMP）.Results:In this study,we found that H₂O₂with a final concentration of 600μmol/L can induce the oxidative damage model of human lens epithelial cell line SRA01/04 cells cultured in vitro,and the oxidative damage became more severe with the increase of H₂O₂concentration and duration.NR at a final concentration of 1mmol/L promoted the proliferation of SRA01/04 cells,and alleviated the inhibition of proliferation of SRA01/04 cells by H₂O₂.Flow cytometry results showed that the apoptosis rates in the control group,model group,and treatment group were 7.30%,44.05%,and 27.16%,respectively.After DCFH-DA dye stained the cells,the fluorescence intensity of green fluorescence seen under the fluorescence microscope reflected the content of ROS in the cells.In the control group,there was no significant green fluorescence.In the model group,the intracellular green fluorescence intensity was significantly increased,indicating that the ROS content was significantly increased,while the green fluorescence intensity of the cells in the treatment group was relatively weakened,which proved that the ROS content was reduced.In the scratch test,the wound healing percentage at 12 hours after scratching of cells in the normal group was 19.84±4.40%,which was significantly higher than that of cells treated with NR（4.74±1.78%,P<0.0001）.Western blot results confirmed that the expression of the anti-apoptotic protein Bcl-2 in the model group was down-regulated,the zymogen form of the apoptotic protein Caspase-3 was down-regulated,and the activated form（Cleaved caspase-3）was up-regulated,which were in accordance with the results of flow cytometry.NR significantly inhibited H₂O₂-induced p-ERK1/2 protein expression in MAPK pathway（P<0.001）.Compared with the control group and the model group,the expression of p-JNK in the treatment group increased,and the difference was statistically significant（P<0.0001,P<0.0001）.In JAK2/Stat3 pathway,compared with the control group,p-JAK2 and p-Stat3 were down-regulated under H₂O₂stimulation.However,compared with the model group,the expression of p-JAK2 and p-Stat3 in the treatment group increased significantly（P<0.001,P<0.0001）.The mitochondria were stained by Mito-Tracker.According to the red fluorescence seen under the fluorescence microscope,the fluorescence intensity of mitochondria in the model group was lower than that in the normal group and the treatment group.Statistical analysis of relative fluorescence intensity showed that compared with the normal group,the MMP in the model group was reduced,and the difference was statistically significant（P<0.05）,while the MMP in the treatment group was significantly higher than the model group（P<0.001）.[Conclusion]:Our finding reveals that NR treatment may alleviated the oxidative damage,inhibited cell apoptosis and migration,and repair mitochondrial damage of SRA01/04 cells induced by H₂O₂through MAPK pathway and JAK2/Stat3 pathway,playing a potential effect on preventing lens from oxidative injury.