Protective effect of rapamycin on hypoxia-injuried retinal ganglion cells and its mechanism

Authors: Ren Jing,  Qin Bo,  Zou Chang,  He Jing,  Liu Shenwen

DOI: 10.3760/cma.j.issn.2095-0160.2017.05.004
Published 2017-05-10
Cite as Chin J Exp Ophthalmol, 2017,35(5): 404-409.

Abstract

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.

Key words:

Rapamycin; Retinal ganglion cells; Mammalian target of rapamycin; Apoptosis

Contributor Information

Ren Jing
Department of Ophthalmology, The First Clinical College of Jinan University, Guangzhou 510632, China
Qin Bo
Shenzhen Eye Hospital Affiliated to Jinan University, Joint College of Optometry of Shenzhen University, Ophthalmic Key Laboratory of Shenzhen, Ocular Trauma Treatment and Stem Cell Differentiation Public Service Platform of Shenzhen, Shenzhen 518040, China
Zou Chang
People’s Hospital of Shenzhen, Clinical Research Centre, Shenzhen 518000, China
He Jing
Shenzhen Eye Hospital Affiliated to Jinan University, Joint College of Optometry of Shenzhen University, Ophthalmic Key Laboratory of Shenzhen, Ocular Trauma Treatment and Stem Cell Differentiation Public Service Platform of Shenzhen, Shenzhen 518040, China
Liu Shenwen
Shenzhen Eye Hospital Affiliated to Jinan University, Joint College of Optometry of Shenzhen University, Ophthalmic Key Laboratory of Shenzhen, Ocular Trauma Treatment and Stem Cell Differentiation Public Service Platform of Shenzhen, Shenzhen 518040, China
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Updated: September 4, 2019 — 12:59 pm