Graefes Arch Clin Exp Ophthalmol (2015) 253:83–90 DOI 10.1007/s00417-014-2771-7

BASIC SCIENCE

Estrogen prevents high-glucose-induced damage of retinal ganglion cells via mitochondrial pathway Ming Hao & Yue Li & Wenjian Lin & Qian Xu & Ning Shao & Yixin Zhang & Hongyu Kuang

Received: 16 December 2013 / Revised: 16 July 2014 / Accepted: 4 August 2014 / Published online: 13 September 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Background Diabetic retinopathy (DR) is a leading cause of acquired blindness in adults. Previous research has shown that the apoptosis of retinal ganglion cells(RGCs) plays an important role in the initiation and development of diabetic retinopathy. The positive effect of estrogen on the nervous system is currently attracting increasing attention. In this study, we investigated whether17β-estradiolum (E2) has protective effects on RGCs in a high-glucose environment. Methods The cell survival rates were measured by Cell Counting Kit-8, the apoptosis was detected by flow cytometry, the intracellular reactive oxygen species (ROS) levels were examined by immunofluorescence method, and the intracellular mitochondrial membrane potential was examined by confocal microscopy. The expression levels of cytochrome C, Bcl-2, and Bax were analyzed by Western blot method. The effect of estrogen receptor blocker tamoxifen on the RGCs was also evaluated. Results It was found that E2 stabilizes the mitochondrial membrane potential, reduces intracellular ROS levels, upregulates Bcl-2 expression, inhibits Bax expression, decreases the generation of cytochrome C, and finally reduces the apoptosis of RGC-5 cells in a high-glucose environment. These protective functions could be attributed to E2 receptors, which make E2 a prospective patent application candidate in the treatment of DR. Furthermore, when cells were pre-cultured with tamoxifen, we found that tamoxifen inhibited the shielding effects of E2.

Ming Hao and Yue Li contributed equally to this work M. Hao : Y. Li : W. Lin : Q. Xu : N. Shao : Y. Zhang : H. Kuang (*) Department of Endocrinology, The First Clinical Hospital of Harbin Medical University, No. 23 Youzheng Street, NanGang District, Harbin, Heilongjiang Province, China e-mail: [email protected]

Conclusion E2 has a broad application prospect in the treatment of DR. Keywords 17β-Estradiolum . Retina ganglion cell . Mitochondrial pathway

Introduction Diabetic retinopathy (DR) is a leading cause of acquired blindness in adults. The rate and incidence of blindness due to DR is rising along with the elevation in diabetic morbidity. Recent research has shown that DR is not only a microvascular disease but also a neural lesion [1, 2]. Retinal ganglion cells (RGCs) are the only efferent neurons that transmit visual information from the retina to the visual centre, and their death could lead to visual field defects and ultimately, blindness. A high glucose level may alter the structure and function of RGCs. It is, therefore, important to pay close attention to the retinal ganglion lesions for early prevention and treatment of DR Estrogen could prevent neurocyte degeneration by acting as a neuroregulator and neuroprotective agent [3]. Recent clinical research has also led to some interesting findings. A study showed that estrogen has protective effects on neurocytes, including RGCs [4]. Another study showed that estrogen is a steroid hormone, whose signal is mediated by the estrogen receptors (ERs). ERs are expressed in the retinal outer plexiform layer, inner nuclear layer, ganglion cell layer, and nerve fiber layer [5]. Epidemiological investigations have shown that the incidence of DR in males with type 2 diabetes is higher than that in females with the condition, and that its occurrence increases with age in females; both these observations indicate that estrogen may be involved in the events leading to DR [6, 7]. At present, more attention has been paid to the positive effect of estrogen on the nervous system [3, 4].

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Mitochondria play an extremely important role in the process of the development of DR. The apoptotic mechanism of RGCs has been investigated in this regard, and more attention has been paid to the effects of mitochondria on apoptosis. As the main energy source of RGCs, mitochondrion is one of the main targets of peroxide. Oxidative stress induces mitochondrial dysfunction, particularly by destroying the mitochondrial membrane potential. This is followed by the destruction of its outer membrane and the release of apoptosis-inducing factors, ultimately leading to apoptosis [8, 9]. Reactive oxygen species (ROS) may promote the release of mitochondrial cytochrome C, which destroys cell membranes and stimulates the occurrence of apoptosis by increasing lipid peroxide level [10, 11]. Some studies have reported that estrogen inhibits the apoptosis of neurons via the mitochondrial pathway in the brain, and that mitochondria are the therapeutic targets [12]. In this study, we established a RGC-5 model of DR in vitro under high-glucose conditions,, explored the protective effects of estrogen on RGCs, and examined the mitochondrial mechanism of the estrogen-mediated protection.

Materials and methods Materials RGC-5 cells were purchased from the cell collection of ATCC. E2 was purchased from the Dr. Ehrenstorfer Company. Tamoxifen, Dulbecco’s modified Eagle’s medium(DMEM), penicillin/streptomycin, fetal bovine serum(FBS) and trypsinEDTA mixture were purchased from Hyclone (Beijing, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technology (Kumamoto, Japan). Reactive Oxygen Species Assay Kit(ROS) and mitochondrial membrane potential assay kit with JC-1(JC-1) were purchased from Beyotime (Jiangsu, China). Annexin V2FITC/PI was purchased from Biosea (Beijing, China). Ripa lysis buffer (RIPA), Phenylmethanesulfonyl fluoride (PMSF) and electrochemiluminescence kit were purchased from Beyotime (Jiangsu, China). Anti-β-action and HRP-labeled Goat AntiMouse IgG(H+L) were purchased from Zhongshang Goldenbridge Biotechonology (Beijing, China).

Cell line culture The RGC-5 cells were cultured in DMEM containing 10 % fetal bovine serum, 25 mM(mol/l) glucose in a humidified atmosphere of 95 % room air and 5 %CO2 at 37 °C as described. These cells had a doubling time of approximately 20 h, and were passaged by trypsin at a ration of 1:9–10 every 3–4 days.

Graefes Arch Clin Exp Ophthalmol (2015) 253:83–90

Cell viability assay RGC-5 in logarithmic growth phase were digested into singlecell suspension with trypsin. 100 μl of cell suspension (5,000 cells/well) was dispensed in a 96-well plate with media containing 25 mM glucose. The plate was pre-incubated for 24 h in a humidified incubator (37 °C, 5 %CO2). Then the cells were switched to DMEM containing 25 mM glucose (control), 55 mM glucose (high-glucose), 55 mM glucose plus E2(10−11,10−10,10−9,10−8,10−7 or 10−6 mol/l respectively). The plate was incubated for 24 h in the incubator, then 10 μl of CCK-8 solution were added into each well of the plate. The plate was incubated for 2 h in the incubator. The absorbance was measured at 450 nm using a microplate reader, and the survival rate of the cells was calculated. The cells were pre-incubated for 24 h (37 °C, 5 %CO2) according to the steps above. Then the cells were switched to DMEM containing 25 mM glucose (control), 25 mM glucose plus 5 μmol/l tamoxifen, 55 mM glucose, 55 mM glucose plus E2, 55 mM glucose plus E2 and 5 μmol/l tamoxifen (the tamoxifen was dissolved in 5 % DMSO). The plate was incubated for 24 h in the incubator, then 10 μl of CCK-8 solution were added into each well of the plate. The plate was incubated for 2 h in the incubator. The absorbance was measured at 450 nm using a microplate reader, and the survival rate of the cells was caculated. The histogram was plotted to show the results: Survival rate (%) = [determination DD/control group DD] ×100 % Cell apoptosis analysis RGCs were trypsinized and adherent cells were collected. Apoptotic cells were evaluated using an Annexin V/FITC kit. Cells were divided into five groups: 25 mM glucose (control), 25 mM glucose plus 5 μmol/l tamoxifen, 55 mM glucose, 55 mM glucose plus E2, and 55 mM glucose plus E2 and 5 μmol/l tamoxifen. Cells were stained according to the manufacturer’s protocol, and analyzed by flow cytometry. Quantitation of ROS Cells were suspended in complete phenolred-free DMEM, and plated in 6-well plates at 5×103 cells/well. The plate was pre-incubated for 24 h in a humidified incubator (37 °C, 5 %CO2). Then the cells were switched to DMEM containing 25 mM glucose (control), 55 mM glucose (high-glucose), 55 mM glucose plus E2, 55 mM glucose plus E2 and 5 μmol/l tamoxifen. The plate was incubated for 24 h in the incubator. Before observed by Immunofluorescence microscopy, cells were washed with PBS, and incubated with DCHF-DA(final concentration 10 μmol/l) for 20 min at 37 °C.

Graefes Arch Clin Exp Ophthalmol (2015) 253:83–90

Measurement of mitochondrial membrane potential One hundred microliters of cell suspension (5,000 cells/well) were dispensed in a 6-well plate with media containing 25 mM glucose. The plate was pre-incubated for 24 h in a humidified incubator (37 °C, 5 %CO2). Then the cells were switched to DMEM containing 25 mM glucose (control), 55 mM glucose (high-glucose), 55 mM glucose plus E2, 55 mM glucose plus E2 and 5 μmol/l tamoxifen. The plate was incubated for 24 h in the incubator. Cells which cultured in six-well plates after indicated treatments were incubated with an equal volume of JC-1 staining solution (5 μg/ml) at 37 °C for 20 min, and rinsed twice with PBS. Cells were then rinsed twice with JC-1 staining buffer. The RGC-5 cells in each treatment group were calculated as the fluorescence ratio of red to green. Western blotting This analysis was performed on 100 μg of protein from each cell lysate. Proteins were blotted onto PVDF membrane after fractionated by SDS–PAGE. The membrane was blocked with 5 % non-fat dried milk at room temperature for 1 h, after which a 1:500 dilution of anti-cytochrome C, anti-Bax, and anti-Bcl-2 were incubated overnight at 4 °C. Then it was washed (three times for 10 min) with TBS. Secondary antibodies (1:5,000) were diluted in blocking solution and incubated with the membranes for 1 h at room temperature. Then they were washed for 30 min (three times for 10 min) with TBS. Finally, the membranes were washed and stained using EC kit. Statistical analysis Data were performed with SPSS11.0 software system and expressed as mean ± SEM. Statistical comparisons were made with Student’s t-test (two treatment groups) or one-way ANOVA test; P

Estrogen prevents high-glucose-induced damage of retinal ganglion cells via mitochondrial pathway.

Diabetic retinopathy (DR) is a leading cause of acquired blindness in adults. Previous research has shown that the apoptosis of retinal ganglion cells...
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