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@#AIM: To investigate the mechanism of Delphinidin(Dp)in protecting retinal against light induced oxidative damage.<p>METHODS: All 661W photosensitive cells were treated with 2 000Lx light(48h)and/or different concentrations of Dp(5, 10, 20μmol/L, 24h). Cell activity, intracellular LDH activity, TBARS content and antioxidant enzymes(SOD, GSH-Px, GST)activity were determined respectively. After the healthy SD rats were treated with 3 000 Lx light(24h)and/or Dp \〖100mg/(kg·d)for 4wk\〗, then changes in retinal tissue structure were observed and fluctuations in oxidative stress index(SOD, GSH-Px, GST)were determined.<p>RESULTS: The results of <i>in vitro</i> experiments showed that the cell activity was significantly decreased after irradiation, the LDH activity and TBARS content were increased, and the activity of antioxidant enzyme system were decreased. However, Dp treatment could increase cell viability, decrease LDH activity and TBARS content, and increase the activity of antioxidant enzyme system. <i>In vivo</i> experiments showed that Dp can protect the structural integrity of retina, reduce the content of TBARS in retinal tissue, and increase the activity of SOD, GSH-Px and GST.<p>CONCLUSION: Dp may protect retinal against Photochemical factors -induced oxidative damage by regulating the oxidation-antioxidant system.
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Objective To construct and authenticate the lentiviral-mediated overexpression of mouse mitochondrial-targeted-8-oxoguanine DNA-glycosylase 1 (mito-OGG1) gene and the lentiviral-mediated short hairpin RNA (shRNA) down-regulation of OGG1 gene expression model in 661W cells.Methods Constructed the target plasmids,including pLenti-EF1a-EGFP-P2A-Puro-CMV-Mito-OGG1-3Flag (pLenti-OGG1-GFP) and pLKD-CMV-G&PR-U6-shRNA (pLKD-shRNA).293T cells were used to obtain green fluorescent protein (GFP)-tagged lentiviral vector of interest by using a second generation lentivirus packaging system.293T cells were also used for the virus titer estimation.The multiplicity of infection (MOI) of 661W cells was detected by fluorescence microscopy.A stable transfected cell line was screened by puromycin.Immunofluorescence was used to detect transfection efficiency and cytochrome C oxidase Ⅳ (COXⅣ)-OGG1 co-localization.OGG1 mRNA and protein expression levels were detected by real-time qantitative PCR (QPCR) and Western blot.Results Sequencing results showed that the inserted sequence in the over-expression plasmid was consistent with the mouse OGG1 (NM_010957.4) gene sequence in the gene library.The original lentiviral titer after packaging and purification was between 2.0× 107to 6.0× 107 TU/ml.The optimal MOI of 661W cells was 40,and puromycin with a concentration of 4.0 μg/ml successfully screened stable transformation.The transfection efficiency was up to 100％ after screening.Immunofluorescence demonstrated successful co-localization of OGG1 and COXⅣ.The relative expression levels of OGG1 mRNA in the blank control group,OGG1 group,overexpression control group,shRNA group and low expression control group were 1.000±0.000,41.581±12.206,0.888±0.056,0.239±0.121 and 1.081±0.083,and the relative expression levels of OGG1 protein were 1.029±0.153,1.657 ± 0.237,0.752 ± 0.143,0.471 ± 0.149 and 1.036 ± 0.185,respectively,with significant differences between them (F=44.654,30.948;both at P＜0.05),the relative expression levels of OGG1 mRNA and protein in the OGG1 group were significantly higher than those in the overexpression control group,the relative expression levels of OGG1 mRNA and protein in the shRNA group were significantly lower than those in the lower expression control group,with significant differences between them (all at P＜0.05).Conclusions The mitoOGG1 overexpression and OGG1 knockdown models of 661W cells are successfully constructed,which provides the preliminary experimental basis for follow-up study.
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Objective To establish the apoptosis model in mouse cone cell line 661W cells and to investigate the viability of 661W cells in the conditions of different levels of autophagy.Methods Different concentrations of anti-Fas antibody were added to establish the apoptosis model of 661W cells,the expression of caspase-3 was detected by Western blot and the appropriate concentration of anti-Fas antibody was screened.Different concentrations of the autophagy inhibitor 3-methyladenine (3-MA) or autophagy inducer rapamycin were added to inhibit or induce autophagy,the expression of microtubule-associated protein 1 light chain 3 (LC-3) Ⅱ/LC-3 Ⅰ were detected by Western blot and the appropriate concentrations were also screened.The cultured cells were divided into 6 groups:control group,simple 3-MA group,simple rapamycin group,model group,model + 3-MA group and model + rapamycin group.Western blot was adopted to detect the expression of caspase-3,caspase-8,autophagy related genes 5 (Atg-5) and LC-3 Ⅱ/LC-3 Ⅰ at 0 hour,3,6,12,24 and 48 hours after induction.Flowcytometer was adopted to detect the apoptosis rate of 661W cells.Results The apoptosis model of 661W cells was successfully established,and the appropriate concentration of anti-Fas antibody was 2.0 μg/ml.After stimulated by the anti-Fas antibody,the expression of caspase-3 and caspase-8 of 661W cells increased from the time point of 6 hours,peaked at 12 hours,and sustained to 48 hours.However,the expression of Atg-5 and LC-3 Ⅱ/LC-3 Ⅰ raised from the time point of 3 hours,peaked at 24 hours,and decreased to the basic level at 48 hours.In addition,the appropriate concentrations of 3-MA and rapamycin were 20 nmol/L and 2.0 nmol/L,respectively.There was no statistical difference among the control group,simple 3-MA group and simple rapamycin group on the expression of caspase-3 and caspase-8 and the apoptosis rate of 661W cells at different time points (all at P＞0.05).The expressions of Atg-5 and LC-3 Ⅱ/LC-3 Ⅰ in the simple rapamycin group were significantly higher than those in the control group at different time points (all at P＜0.05).The expressions of caspase-3 and caspase-8 and the apoptosis rate in the model + 3-MA group were significantlly higher than those in the model group at 3,6,12,24 and 48 hours after induction,while the expressions of Atg-5 and LC-3 Ⅱ/LC-3 Ⅰ were obviously lower than those in the model group at 3,6,12 and 24 hours after induction (all at P＜0.05).The expressions of caspase-3 and caspase-8 and the apoptosis rate at 6,12,24 and 48 hours after induction in the model+rapamycin group were significantly lower than those in the model group,while the expressions of Atg-5 and LC-3 Ⅱ/LC-3 Ⅰ at the time points of 3,6,12 and 24 hours after induction were obviously higher than those in the model group (all at P ＜ 0.05).Conclusions Under the condition of anti Fas antibody inducing apoptosis,enhancing autophagy can reduce the apoptosis rate of cells,inhibiting autophagy can increase the apoptosis rate.Autophagy may play a protective role in 661W cells.
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Aim To investigate the possible effect of N-acetyl-leu-leu-norleucinal (ALLN),an inhibitor of Calpain,on H2O2-induced damage in 661W cells.Methods The cellular survival of 661W treated with different doses of H2O2 without or with ALLN for 24 h was measured by MTT assay.The protein level of Calpain 1 and Calpain 2 was assessed by Western blot.Results Upon the H2O2 treatment at the concentrations of 50,100,500,1 000 μmol · L-1,the survival rate of 661W significantly decreased in a dose-dependent manner compared to that of the control group.Furthermore,the protein level of Calpain 1 and Calpain 2 showed an obviously time-dependent increase in 661W cells treated with 100 μmol · L-1 H2O2 for 12,18,24 h.Finally,the survival rate of 661W treated with ALLN and H2O2 was higher than that treated only with H2O2,and there was no difference in survival rate between ALLN groups (at the concentrations of 25,50,100,200 μmol · L-1) and control group.Conclusions Calpain is involved in the damage induced by H2O2.ALLN,the inhibitor of Calpain,attenuates the oxidative damage,which plays a promising protective role in photo-receptor cells under oxidative stress.