Regulation Of Oxidative Stress By Methionine Sulfoxide Reductases In Lens

Diabetes and its complications, which were closely related with oxidative stress, areserious diseases that affect on human health. Methionine (Met) is one of the most easilyoxided amino acids, and its main oxidation product is methionine sulfoxide (MetO). Inserious cataract, it has been detected that45%Met residues were modified to MetO.Fortunately, methionine sulfoxide reductases (Msrs) can catalyze MetO derivatives to Met;among all of the Msrs, MsrB1is the only selenoprotein. Previous researches have provedMsrs play important role in maintaining cell viability, but their roles in diabetic cataractdevelopment are still unclear.Here, we used Streptozocin (STZ)-induced diabetic mice and MsrB1RNAinterference in human lens epithelial cell line SRA01/04(HLECs) to observe Msrsexpressions in mouse lens and HLECs, and specially investigate MsrB1role in protectinglens cells against oxidative injury and inhibiting apoptosis. The main results are as below:(1) The related biochemical parameters in STZ-induced diabetic mice after10-dayfeeding were detected by optical microscope, native PAGE, RT-PCR, real-time PCR,western blot, HPLC, TBA assay, DTNB assay and DNPH assay. In diabetic mouse lens,although there were no significant morphological alterations, the total protein componentsby native PAGE revealed that a band around40kDa was visibly decreased, and a bandaround55kDa was increased, the mRNA expression levels of the four Msrs and theprotein expression level of MsrB1decreased, the levels of MetO, malondiadehyde (MDA),protein carbonyl (PC) were all increased accompany with a decrease on the level of totalsulfhydryl (TSH), indicating the increasing oxidative injurie and the decline ofantioxidative capability of diabetic mouse lens. These results suggest that STZ not onlycauses increased oxidative stress, but also suppresses Msr mRNA and MsrB1proteinexpression during early-stage diabetes in mice. (2) MTT assay, real-time PCR, western blot, fluorescent spectrophotometry, TBAassay, fluorescent microscopy, flow cytometry, ELISA and colorimetry were used for inhigh-glucose-induced or MsrB1-gene-silenced HLECs. The viabilities of MsrB1-gene-silenced and high-glucose-induced cells were all decreased; high glucose exposuresignificantly decreased the mRNA and protein expression levels of MsrB1; reactiveoxygen species (ROS) and MDA levels were significantly increased inMsrB1-gene-silenced and high-glucose-induced cells; mitochondria membrane potentialwas markedly decreased, and release of cytochrome c to cytoplasm, caspase-3activity andapoptosis rate were significantly increased, in the MsrB1gene silent or/and high glucoseexposed cells; besides, morphological observation by Hoechst also showed the sameresults. All of the results indicated that MsrB1plays important roles in protecting HLEcell mitochondria against oxidative damage and inhibits oxidative stress-induced apoptosisin diabetic cataracts by scavenging ROS.(3) The related biochemical parameters in sodium-selenite-pretreatment high-glucose-induced or MsrB1-gene-silenced HLECs were detected by transmission electronmicroscope (TEM), real-time PCR, western blot, fluorescent spectrophotometry, TBAassay, ELISA and fluorescent microscopy. The results showed that pretreatment ofsodium-selenite seem to maintain the normal morphology of mitochondria after highglucose or MsrB1gene silent treatments, reduced the ROS, MDA, cytochrome c releaselevels and the number of apoptotic bodies in HLECs; besides, the increased MsrB1expression levels, suggesting sodium-selenite seems to increase the cell viability andantioxidative capability of HLECs by increasing MsrB1and other selenoproteinsexpressions.