| Purpose:Age-related cataract(ARC)is the leading cause of blindness worldwide,which is related to the apoptosis of lens epithelail cells(LECs)resulted from excessive reactive oxygen species(ROS)accumulation and mitochondrial dysfunction.It is well accepted that high concentrations of hydrogen peroxide(H2O2)can lead to the opacification of lens and the apoptosis of LECs in vivo and in vitro.Recently,carbon monoxide,one of the by-products of heme-oxygenase(HO),is recognized as a gasotransmitter of anti-inflammatory,anti-oxidative,and anti-apoptitic properties,however,little is known about its effects on LECs.On the basis of these recent studies,carbon monoxide releasing molecule-3(CORM-3),which is water soluble and can simulate the physiological conditions of CO,was used for investigating whether CO can protect rabbit LECs from H2O2-induced apoptosis and its effect on modulating mitochondrial biogenesis,enegy metabolism,and mitochondrial apoptosis cascade.Methods:The eye samples were extracted from New Zealand rabbits(2.0-2.5 kg,6 months old),and the anterior capsules were obtained from the lens and cultured in culture media.LECs were identified by immunofluorescence staining using anti-alpha B-crystallin antibody(1:200).We used CCK-8 to determine the optimal concentration and incubation time of H2O2,CORM-3,and iCORM-3.Deoxy-myoglobin(deoxy-Mb)was used for measuring the release of CO from CORM-3.A H2O2 assay kit was used to determine H2O2 concentrations in culture media with or without CORM-3 and iCORM-3.The nuclear translocation of nuclear factor-kappa B(NF-?B)p65 was monitored by Western blot and immunofluorescence staining.In addition,the levels of intracellular ROS,antioxidants,and apoptotic molecules(Bax,Bcl-2 and caspase-3)were measured respectively.Furthermore,cell apoptosis rate was quantified by flow cytometry.Mitochondria,cystolic protein,and mitochondrial protein were obtained from rabbit LECs by mitochondria isolation kit.To evaluate mitochondrial biogenesis,several mitochondrial transcription factors(PGC-1?,NRF-1,and mtTFA)were detected by western blot analysis.To assess cellular metabolism,adenosine triphosphate(ATP)levels and mitochondrial cytochrome c oxidase(COX)enzymatic activity were measured.In addition,mitochondrial permeability transition pores(mPTP)opening,dissipation of mitochondrial membrane potential(??m),cytochrome c mitochondrial translocation,and apoptotic molecules were also detected to evaluate mitochondrial apoptosis pathway.Furthermore,the interaction of Bcl-2 and COX was assessed by co-immunoprecipitation.Finally,CO-mediated regulation of cellular function was detected in Bcl-2-knockdown cells.Data were analyzed with SPSS version 22.0 software,and expressed as the mean ± SD.Groups differences were calculated using analysis of variance(ANOVA)with Bonferroni's multiple comparison test,and P<0.05 was considered statistically significant.Results:One mole of CORM-3 generated almost 1 mol of MbCO,which in turn represents 1 mol of CO liberation.A decrease of relative cell viability occurred in the high concentrations of CORM-3(100 and 200 ?M).Cells pretreated with 50 ?gM CORM-3 for 6 h showed greater viability than those in the control group(P<0.001),thus we selected a 6 h pretreatment with 50 ?M CORM-3 before exposure to 400?M H2O2.The H2O2 concentrations gradually decreased with time.This did not change in the presence of either 50 ?M CORM-3 or iCORM-3 when mixed with 400 ?M H2O2 for different periods.These data suggest that the effect of CO was specific to LECs protection.Low concentrations of CO released from CORM-3 can attenuate NF-?B p65 nuclear translocation,reduce ROS generation and enhance intracellular glutathione and superoxide dismutase levels.Moreover,low concentrations of CO inhibited H2O2-induced apoptotic molecules,thereby decreasing the apoptosis of LECs.Low concentrations of CO pretreatment restored H2O2-induced down-regulation of mitochondrial transcription factors expression,COX activity and ATP production.Moreover,CO pretreatment attenuated mPTP opening,??m loss,cytochrome c mitochondrial translocation,and activation of apoptotic molecules.CO pretreatment was identified to enhance the interaction of Bcl-2 with COX,and silence of Bcl-2 expression prevented CO-regulated cellular metabolism and cytoprotection.Conclusions:Low concentrations of CO protect LECs from H2O2-induced oxidative damage by attenuating NF-?B p65 nuclear translocation,reducing the generation of ROS and apoptotic molecules,and restoring antioxidant enzyme levels,thereby inhibiting LECs apoptosis.CO modulates H2O2-induced cellular dysfunction by increasing mitochondrial biogenesis,enhancing cellular metabolism,and attenuating mitochondrial apoptosis cascade.Moreover,Bcl-2 expression was vital for CO to regulate cellular metabolism and cytoprotection in LECs. |
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