Protective Action And Mechanism Of GBE And Sulforaphen On The Apoptosis Of Retinal Nerve Cells In Mice

Objective:Light can cause retinal cell apoptosis and induce retinal outer nuclear layer impairment. In this study, the protective action and mechanism of GBE and SF on light-induced retinal cell apoptosis is discussed by GBE and sulforaphane (SF) heart injection on retinal light damaged mice.Method:1. Establish mouse model of light damage:Bal b/c mice were bred in dark room for 24 hours and placed in light boxes the next day. The mice were killed respectively at 24h,48h and 72h with light exposure, the eyes were made into paraffin sections. HE staining was conducted for retinal morphology; there were outer nuclear layer thickness measurement and outer nuclear layer number statistics for the analysis of retinal damage with different length of light;DNA gradient gel electrophoresis and TUNEL assay were employed for the measurement of cell apoptosis.2. Bal b/c mice were randomly divided into 6 groups as normal control group, experimental control group (saline), GBE experimental group(GBE-Ⅰand GBE-Ⅱ), SF experimental group(SF-Ⅰand SF-Ⅱ), n=5, with cardiac injection (instead of intravenous). Except the normal control group, mice in the other three groups were under light exposure in the home-made light box and sacrificed 72h later, routine eyeball paraffin sections were prepared, there were morphological observation after HE staining and measurement for the thickness of the outer nuclear layer; DNA gradient gel electrophoresis and TUNEL assay for retinal cell apoptosis; RT-PCR for the expression of cytochrome C and Bak-1; Caspase activity kit for Caspase-3 activity. SPSS 13.0 statistical software was used for single factor of variance analysis.Results:1. Mouse model with light damage:retinal structure levels of mice in normal group was clear through morphological observation with HE staining; outer nuclear layer was significantly thinner in each group with light exposure, the inner and outer segments of retinal visual cells were arranged in disorder, with unclear boundaries and irregular nuclei. Apoptosis detection and localization:DNA electrophoresis showed that DNA laddering was present in each group with light exposure, among which DNA ladder in the group with 72h light exposure was the most significant; TUNEL assay showed that apoptosis was in the outer nuclear layer.2. Preventive effect of GBE and SF on retinal light damage:It was morphologically observed that retinal cells were sparsely arranged in experimental control group, the inner and outer sections were disorderly arranged, swelling and broken, the outer nuclear layer was significantly thinner; retinal structure of the control group and dose group was better than that in experimental control group. Measurements of outer nuclear layer thickness:normal control group was 49.23±2.32μm, experimental control group was 40.36±2.17μm; outer nuclear layer thickness of GBE-Ⅰand GBE-Ⅱgroups were 42.0770±0.8999μm and 40.9411±0.7060μm; outer nuclear layer thickness of SF-Ⅰand SF-Ⅱgroups were 48.2352±2.0447μm and 46.8004±0.6014μm. Statistical analysis showed that the treatment group (GBE-Ⅰgroup, GBE-Ⅱgroup, SF-Ⅰgroup, SF-Ⅱgroup) was significantly different (P<0.001) compared with experimental control group.3. The prevention and protection mechanism of GBE and SF on retinal light damage:In GBE group(GBE-Ⅰgroup,GBE-Ⅱgroup), SF experimental group(SF-Ⅰgroup,SF-Ⅱgroup) and control group, RT-PCR results showed that the expression of cytochrome C and Bak-1 were reduced to varying degrees. The expression levels of mRNA in SF group(SF-Ⅰgroup,SF-Ⅱgroup), GBE group(GBE-Ⅰgroup,GBE-Ⅱgroup) were significantly different compared with control group (P<0.01, P<0.05); Test results of Caspase-3 activity:Caspase-3 activity in SF group (SF-Ⅰgroup,SF-Ⅱgroup) and the GBE group(GBE-Ⅰgroup,GBE-Ⅱgroup) was both reduced compared with the experimental control group.Conclusion:1. mouse model with retinal light damage can be established through regular light exposure;2. GBE and SF have preventive effects on retinal light injury;3. The prevention and protective mechanisms of GBE and SF on retinal light damage were associated with the inhibition of retinal cell apoptosis and the related down-regulation of protein expression.