Role Of MsrB1(SeIR) In Cytoprotection Against Peroxynitrite-Induced Damage In Human Lens Epithelial Cells

Cataract is a visible opacity in the lens substance, which poses a substantial economic and public health burden and is the leading cause of blindness worldwide. Peroxynitrite (ONOO-) is the reaction product of superoxide (O2) and nitric oxide (NO). The molecule rapidly breaks down at physiological pH to yield the hydroxyl radical (·OH) and nitrogen dioxide radical (·NO2). As a potent oxidant and nitrating species, ONOO-is proposed to be an effector of cell damage in selenite cataract and diabetic cataract, by oxidizing and/or nitrating biological molecules including proteins, lipids, and DNA. Previous studies have proved that ONOO-can oxidize Met residues of proteins to MetO, and the formation of nitrotyrosine is considered a footprint of oxidative damage mediated by ONOO-. Other selenoproteins, such as glutathione peroxidases, thioredoxin reductase and selenoprotein P have been shown to play a potential role in protection against ONOO-. However, the role of MsrB1, which is a selenoprotein named selenoprotein R, in cellular protection against ONOO--induced hLE cells damage remains to be evaluated, and a role in regulating certain cell signaling molecules and certain proteins remain unclear. So the role of MsrBl in cataract formation and development needs a further study.In this paper, the role and mechanism of MsrB1in protection against ONOO--induced hLE cells damage were investigated. The main results are as follows:(1) Involvement of MsrBl in the regulation of redox balance and inhibition of peroxynitrite-induced apoptosis in hLE cells.In an attempt to shed light on the roles of MsrBl, known as selenoprotein R, in protecting hLE cells against peroxynitrite damage, and contribution of loss of its normal activity to cataract, the influences of MsrB1gene silencing on peroxynitrite-induced apoptosis in hLE cells were studied by real time RT-PCR analysis, MTT assay, Western blotting, transmission electron microscopy, fluorescence microscopy, flow cytometry, ROS and MDA measurement kit. The results showed that low concentrations of ONOO-markedly stimulated the proliferation of hLE cells, while the viabilities of hLE cells treated with high concentrations of ONOO-were significantly decreased; both exogenous peroxynitrite and MsrB1gene silencing by short interfering RN A (siRNA) independently resulted in oxidative stress, endoplasmic reticulum (ER) stress, activation of caspase-3as well as an increase of apoptosis in hLE cells; moreover, when MsrB1-gene-silenced cells were exposed to300μM peroxynitrite, these indexes were further aggravated at the same conditions and DNA strand breaks occurred. The results demonstrate that in hLE cells MsrB1may play important roles in regulating redox balance and mitigating ER stress as induced by oxidative stress under physiological conditions; MsrB1may also protect hLE cells against peroxynitrite-induced apoptosis by inhibiting the activation of caspase-3and oxidative damage of DNA under pathological conditions. Our results imply that loss of its normal activity is likely to contribute to cataract.(2) MsrB1protects human lens epithelial cells against peroxynitrite-induced F-actin disruption.Previous studies have demonstrated that MsrBl can resist to oxidative stress and may act as a ROS scavenger, as well as have a potential role in protection against ONOO-. In the present study, the role of MsrB1in protecting hLE cells against ONOO--induced F-actin disruption was studied by Western blotting, immunoprecipitation, U0126inhibitor treatment and immunofluorescence. The results showed that low concentrations of ONOO-might induce assembly of F-actin, while high concentrations of ONOO-might diminish assembly of F-actin, and when MsrB1-gene-silenced hLE cells were exposed to ONOO-, F-actin protein levels were further decreased; Low concentrations of ONOO activated ERK1/2and high concentrations of ONOO-inhibited ERK1/2activation, which was consistent with the changes of assembly or disassembly of F-actin by low or high concentrations of ONOO-; The results of immunoprecipitation showed that disruption of F-actin was accompanied by increase of F-actin nitration. Furthermore, bFGF-activated ERK1/2phosphorylation occurs via a MEK-dependent pathway, whereas ONOO--activated ERK1/2phosphorylation occurs via a MEK-independent pathway. In summary, both high concentrations of ONOO and MsrB1gene silencing by siRNA independently increased disassembly of F-actin and induced inactivation of ERK in hLE cells; moreover, MsrB1gene silencing could significantly aggravate disassembly of F-actin by ONOO-through increase of nitration of F-actin and inactivation of ERK, suggesting that MsrB1may play important roles in protecting hLE cells against ONOO--induced F-actin disruption by inhibiting nitration of F-actin and inactivation of ERK. These results imply that loss of its normal activity is likely to influence the lens cell elongation and differentiation.(3) Role of MsrBl in cytoprotection against BCS-induced oxidative stress in hLE cells.In the present study, the role of MsrB1in protecting hLE cells against BCS-induced apoptosis and oxidative stress were studied by MTT assay, Cu/Zn-SOD and ROS measurement kit, fluorescence microscopy and Western blotting. The results showed that both BCS (1000μM) and MsrB1gene silencing by short interfering RNA (siRNA) independently resulted in oxidative stress, accumulation of ROS levels and an increase of apoptosis in hLE cells; moreover, when MsrB1-gene-silenced cells were exposed to1000μM BCS, ROS levels and cell apoptosis were further aggravated at the same conditions. Whereas the Cu/Zn-SOD activity in MsrB1-gene-silenced cells was significantly increased, implying that MsrBl gene silencing by siRNA induces an increase of ROS levels and than stimulate the increase of Cu/Zn-SOD activity. Moreover, the results of Western blotting showed that BCS and MsrB1gene silencing induced hLE cell apoptosis accompanied by the inhibition of ERK1/2activation, since the formation of ONOO-induced ERK nitration. In addition, the protein nitration detection results also confirmed the generation of ONOO-. In summary, both BCS and MsrBl gene silencing resulted increase of apoptosis in hLE cells by accumulation of ROS levels and inhibition of ERK1/2activation. Our results suggest that MsrBl may protect hLE cells against BCS-induced apoptosis by regulation of ERK1/2phosphorylation and nitration via scavenging ROS directly or indirectly.