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AIM: To construct an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R) induced injury to the optic nerve and to study the oxidative damage mechanism of ischemia-reperfusion (I/R) injury in 661W cells and the protective effect of ginsenoside Rg1. METHODS: The 661W cells were treated with different concentrations of Na2S2O4 to establish OGD/R model in vitro. Apoptosis, intracellular reactive oxygen species (ROS) levels and superoxide dismutase (SOD) levels were measured at different time points during the reperfusion injury process. The injury model was pretreated with graded concentrations of ginsenoside Rg1. Real-time polymerase chain reaction (PCR) was used to measure the expression levels of cytochrome C (cyt C)/B-cell lymphoma-2 (Bcl2)/Bcl2 associated protein X (Bax), heme oxygenase-1 (HO-1), caspase9, nuclear factor erythroid 2-related factor 2 (nrf2), kelch-like ECH-associated protein 1 (keap1) and other genes. Western blot was used to detect the expression of nrf2, phosphorylated nrf2 (pnrf2) and keap1 protein levels. RESULTS: Compared to the untreated group, the cell activity of 661W cells treated with Na2S2O4 for 6 and 8h decreased (P<0.01). Additionally, the ROS content increased and SOD levels decreased significantly (P<0.01). In contrast, treatment with ginsenoside Rg1 reversed the cell viability and SOD levels in comparison to the Na2S2O4 treated group (P<0.01). Moreover, Rg1 reduced the levels of caspase3, caspase9, and cytC, while increasing the Bcl2/Bax level. These differences were all statistically significant (P<0.05). Western blot analysis showed no significant difference in the protein expression levels of keap1 and nrf2 with Rg1 treatment, however, Rg1 significantly increased the ratio of pnrf2/nrf2 protein expression compared to the Na2S2O4 treated group (P<0.001). CONCLUSION: The OGD/R process is induced in 661W cells using Na2S2O4. Rg1 inhibits OGD/R-induced oxidative damage and alleviates the extent of apoptosis in 661W cells through the keap1/nrf2 pathway. These results suggest a potential protective effect of Rg1 against retinal I/R injury.
RESUMO
OBJECTIVE: To investigate the existence and distribution characteristics of neural stem cells in the eyes of adult human ciliary body and retina. METHODS: Eight eyes from 20 - 40 years old health adult and 3 infant eyes were obtained from Guangdong Eye Bank and were used in the present studies. The protein and mRNA expressions of neural progenitor cell-specific antigen nestin in the ciliary body and retina were detected by immunohistochemical staining and reverse RT-PCR assays, respectively. The ultrastructural and distribution characteristics of these cells were studied by transmission electron microscopy (TEM). RESULTS: Immunohistochemical staining showed that there were a few nestin-positive cells existed in pigment epithelium layer of ciliary body and inner nuclear layer (INL) of retina. mRNA expression of nestin gene was detected in both the ciliary body and retina of the infant and adult eyes by RT-PCR assays. The expression of nestin in the infant ciliary body was higher than that of the adult eye. By TEM, a few cells with the characteristic morphology of neural stem cells were identified and their location was consistent with the results of immunohistochemical staining. CONCLUSION: Cells with the features of neural stem cells are present in the pigment epithelium layer of ciliary body and the inner nuclear layer of retina in adult human eyes.