Authors: Ren Jing, Qin Bo, Zou Chang, He Jing, Liu Shenwen
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Background
Studies showed that rapamycin (Rapa) plays a protective effect on central nervous system by improving the aging of human brain tissue and ameliorating rat cerebral metabolism after ischemia.Optical nerve and retinal ganglion cells (RGCs) are central nerve, however, whether Rapa can protect hypoxia-injuried RGCs is still unclear.
Objective
This study was to explore the protective role of Rapa on hypoxia RGC-5, a rat RGC line, and its underlying mechanism in order to provide a new strategy for the treatment of traumatic optic neuropathy.
Methods
Rat RGC-5 cells were cultured using DMEM with 10% fetal bovine serum, and the cells were observed under the inverted phase contrast microscope.The cells were treated by 50, 100, 200, 400 and 600 μmol/L CoCl2 for 24 hours and 48 hours, respectively, and Clone Select Imager was employed to assess the survival rate.The CoCl2-induced hypoxia cell models were established by adding 200 μmol/L CoCl2 in the medium for 24 hours, and then the 0.1, 0.4, 1.6, 6.4 μmol/L Rapa was used to treat the models for 24 hours in the Rapa intervention group, respectively.The cells cultured by DMEM with 10% fetal bovine serum served as the normal control group.The survival rate of the cells was evaluated by Clone Select Imager; the apoptotic rate of the cells was assayed by AnnexinV-FITC/PI double-staining flow cytometry; JC-1 probe was used to detect the mitochondrial trans-membrane potential, and the expression of bax mRNA in the cells was detected by real-time fluorescence quantitative PCR.
Results
The survival rate of the cells was (70.51±5.00)% in the 200 μmol/L CoCl2-treated group, which was significantly lower than (100.00±3.29)% in the normal control group (P<0.01). The survival rate of the cells was significantly different among the normal control group and 0.1, 0.4, 1.6, 6.4 μmol/L Rapa intervention groups (F=167.904, P=0.000), and the survival rate was evidently higher in the 0.1 μmol/L Rapa intervention group than that in the model control group (P<0.05). The apoptotic rate was 25.4%, 37.7% and 25.3%, while mitochondrial trans-membrane potential reduced by 0.4%, 6.3% and 1.4% in the normal control group, model control group and 0.1 μmol/L Rapa intervention group, respectively.The relative expression of bax mRNA in the cells was 1.01±0.21, 3.52±0.30 and 1.66±0.20 in the normal control group, model control group and 0.1 μmol/L Rapa intervention group, showing a significant difference among the groups (F=88.034, P=0.000), and the relative expression of bax mRNA in the model control group was considerably elavated in comparison with the normal control group and 0.1 μmol/L Rapa intervention group (both at P<0.05).
Conclusions
Rapa protects the RGC-5 cells against CoCl2-induced hypoxic damage primarily by down-regulating the expression of bax in the cells and improving the survival rate of RGC-5 cells in vitro.