Protective effect of extract of ginkgo biloba on oxidative damage of human Müller cells

Authors: Sun Yanyan,  Yang Xu,  An Huijuan,  Li Qingfu,  Bao Yuzhou
DOI: 10.3760/cma.j.issn.2095-0160.2015.09.008
Published 2015-09-10
Cite as Chin J Exp Ophthalmol, 2015,33(9): 805-810.

Abstract                              [Download PDF] [Read Full Text

Background

Oxidative stress is a pathophysiological process of retina, so it is very important to explore a protective way against retinal oxidative stress. Studies determined that extract of ginkgo biloba (EGb) has antioxidant, anti-apoptosis, anti-thrombosis and anti-inflammatory effects, however, the effect of EGb on human Müller cells in oxidative stress is still below understood.

Objective

This study was to investigate the protection of EGb against oxidative stress of human retinal Müller cells induced by As2O3in vitro.

Methods

Human retinal Müller cell line was cultured in DMEM/F12 with 10% fetal bovine serum (FBS), 2 μmol/L glutamine and 1% antibiotics. As2O3 solution at the final concentration 5 mg/L was added in the medium for 24 hours to establish oxidative models, and then the EGb with the final concentrations of 5, 10 and 20 mg/L was used to cell models for 24 hours, respectively. Cell viability was detected by MTT assay, and reactive oxygen species (ROS) levels in the cells were detected with CM-H2DCFDA fluorescent probe. The relative expression levels of caspase-3 mRNA in cytoplasm and cell nuclei were assayed by reverse-transcription PCR (RT-PCR), and the expressions of Nrf2 protein were quantitatively detected by Western blot.

Results

Müller cells adhered well 24 hours after cultured. At 6-7 days after culture, Müller cell body was large with abundant cytoplasm and mosaic-like arrangement. However, floating cells were seen after As2O3 treatment. Cell viability (absorbance) was significantly different among the normal culture group, As2O3-treated group, As2O3+ 5 mg/L EGb group, As2O3+ 10 mg/L EGb group and As2O3+ 20 mg/L EGb group, with the strongest viability in the normal culture group and the weakest viability in the As2O3-treated groups (F=163. 57, P=0. 00). The fluorescence intensity of ROS was the weakest in the normal culture group and the strongest in the As2O3-treated group and was gradually weakened with the increase of EGb doses, showing a remarkable difference among the groups (F=4 013. 61, P=0. 00). The relative expression level of caspase-3 mRNA in the cells was gradually reduced with the increase of EGb doses, with a statistically significant difference among the groups (F=2 199. 72, P=0. 00). In addition, no considerable difference was seen in the expression level of Nrf2 protein (grey scale) in cytoplasm among the groups (F=15. 42, P=0. 40); while in the nuclei, the expression levels of Nrf2 protein were 100. 01±0. 04, 46. 59±0. 63, 54. 51±0. 62, 59. 93±0. 17 and 67. 60±0. 24 in the normal culture group, As2O3-treated group and As2O3+ 5 mg/L EGb group, As2O3+ 10 mg/L EGb group, As2O3+ 20 mg/L EGb group respectively, with a significant difference among them (F=7 271. 72, P=0. 00).

Conclusions

EGb can protect human retinal Müller cells against As2O3-induced damage in a dose-dependant manner by antioxidant and antiapoptotic effects in vitro, and the activities occur primarily in cell nucleus.

Key words:

Ginkgo biloba; Plant extracts/pharmacology; Cell line; Müller cells/drug effects; Cell hypoxia; Oxidative stress; Cell survival/drug effects; Apoptosis/drug effects; Humans

Contributor Information

Sun Yanyan
Henan University of Traditional Chinese Medicine, Zhengzhou 450008, China
Yang Xu
An Huijuan
Li Qingfu
Bao Yuzhou
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