Cell Biochem Biophys (2015) 71:749–755 DOI 10.1007/s12013-014-0259-z

ORIGINAL PAPER

Erythropoietin (EPO) Protects Against High Glucose-Induced Apoptosis in Retinal Ganglional Cells Yunxiao Wang • Hui Zhang • Yanping Liu Ping Li • Zhihong Cao • Yu Cao



Published online: 7 October 2014 Ó Springer Science+Business Media New York 2014

Abstract The aim of this study was to investigate the protective effect and mechanism of EPO on the apoptosis induced by high levels of glucose in retinal ganglial cells (RGCs). High glucose-induced apoptosis model was established in RGCs isolated from SD rats (1–3 days old) and identified with Thy1.1 mAb and MAP-2 pAb. The apoptosis was determined by Hochest assay. The levels of ROS were quantitated by staining the cells with dichloro-dihydro-fluorescein diacetate (DCFH-DA) and measure by flow cytometry. The SOD, GSH-Px, CAT activities, and levels of T-AOC and MDA were determined by ELISA. Change in mitochondrial membrane potential (Dwm) was also assessed by flow cytometry, and expressions of Bcl-2, Bax, caspase-3, caspase-9, and cytochrome C were assessed by western blotting. The RGCs treated with high glucose levels exhibited significantly increased apoptotic rate and concentrations of ROS and MDA. Pretreatment of the cells with EPO caused a significant blockade of the high glucose-induced increase in ROS and MDA levels and apoptotic rate. EPO also increased the activities of SOD, GSH-Px, and CAT, and recovered the levels of T-AOC levels. As a consequence, the mitochondrial membrane potential was improved and Cyt c release into the cytoplasm was prevented which led to significantly suppressed up-regulation of Bax reducing the Bax/Bcl-2 ratio. The expressions of caspase-3 and caspase-9 induced by high glucose exposure were also ameliorated in the RGCs treated with EPO. The protective effect of EPO against apoptosis was mediated through its antioxidant action. Thus, it blocked the generation of pro-apoptotic proteins and apoptotic degeneration of the RGCs by preventing the mitochondrial damage. Y. Wang  H. Zhang  Y. Liu  P. Li  Z. Cao  Y. Cao (&) The Affiliated Hospital of Qingdao University, Qingdao 266003, China e-mail: [email protected]

Keywords Oxidative stress  Erythropoietin  Retinal ganglial cells  Mitochondrion pathway of apoptosis

Introduction Diabetic retinopathy (DR) is one of the most common and serious microvascular complications of diabetes mellitus (DM) that is vision threatening. The risk of DR increases with the course of DM. Thus, the risk of DR has been indicated to be 25 % in 5 years, and it increases to 60 % in 10 years and up to 75–80 % in 15 years [1]. Recent studies have shown that apoptosis of retinal ganglial cells (RGCs) is an important sign detected early in DR, which proceeds to change in retinal capillaries leading finally to retinal microangiopathy [2, 3]. Erythropoietin (EPO) has been found to play a protective role against hypoxia, which caused damage to the brain [4]. The in vivo animal studies have also shown the EPO-mediated protection against retinal vascular and neuronal damage [5]. An involvement of overproduction of ROS, oxidative stress, and damage of mitochondrial membrane in the pathogenesis of DR has been recognized [6, 7]. EPO has been indicated to enhance the antioxidant capacity by activating the ROS neutralizing enzymes, SOD, GSH-Px, and CAT [8–10]. In this study, we investigated the EPO-mediated protective effect and mechanism of its action against apoptosis of the retinal ganglial cells (RGCs) induced by high glucose.

Materials and Methods Animals and Reagents SPF Grade Wistar Rats (1–3 days old) were obtained from the Experimental Animal Center of Qingdao Food and

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Drug Administration (Qingdao, Shandong, China). EPO, 5-Bromouracil deoxyriboside (5-Brd), and MTT were purchased from Sigma (St. Louis, MO, USA). Primary antibodies against Thy1.1 and Map2 were purchased from Abcam. TRITC-IgG and FITC-IgG were purchased from Beijing Zhong Shan -Golden Bridge Biological Technology CO., LTD (Beijing, China). DMDM/F12 culture medium was purchased from Gibco. FBS and trypsin were purchased from Hyclone. ROS detection kit, acetyl-L-cysteine (NAC), was purchased from Beyotime Company (Shanghai, China). Detection kits for SOD, GPH.Px, CAT, T-AOC, and MDA were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). All other chemicals were of analytic grade. Culture and Purification of Primary RGCs The culture flasks and plates were coated with poly-L-lysine (100 lg/ml) and dried before use. Wistar rats (1–3 days old) were immersed in 75 % alcohol for 5 min, and eye ball was removed under sterile conditions. Stratum neuroepitheliale retinae was separated with blunt dissection and sheared. Then, the tissue was digested with 0.125 % trypsin and centrifuged for 15 min. The cell pallet was resuspended in DMEM/F12 culture medium containing 10 % serum at a density of 1 9 106/ml. Cells were maintained at 37 °C for 24 h before 5-Bromouracil deoxyriboside (final concentration 20 lg/ml) to inhibit the growth of non-neural cells. Culture medium was changed after 48 h and then every 2–3 days.

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Establishment of High Glucose Model Following the treatment with various concentrations of glucose, trypan blue exclusion assay was performed to assess the viability of cells. Based on the results, 30 mmol/ L treatment for 48 h was chosen to establish high glucose model. The cells were treated as Untreated (control), EPO (20 U/ml EPO), HG (30 mM glucose), HG ? 10 U/ml EPO (30 mM HG ? 10 U/ml EPO), HG ? 20 U/ml EPO, HG ? 40 U/ml EPO, and HG ? 5 mmol/L NAC (Nacetyl-L-cysteine). Apoptosis Measurement by Hoechst Staining Cells were seeded at a density of 5 9 105/ml in poly-Llysine-coated 6-well plates and allowed to grow with glucose until they were 80 % confluent. Then, they were treated with different concentrations of EPU and NAC for 48 h. The cells were then stained according to the manufacturer’s instructions. In short, culture medium was removed, and cells were incubated with 1 ml of fixing solution for 10 min, and then washed twice with PBS. Hoechst 33258 (2 lg/ml, 0.5 ml) was added to the cultures and incubated for 30 min at 37 °C. The stained cells were examined using a fluorescent microscope, and 500 cells were counted in three randomly chosen fields to determine the apoptosis. Measurement of Change in Mitochondrial Membrane Potential (MMP), Dwm

Identification of RGCs The cells grown on slide cover glass for 5 days were washed 3 times with PBS for 5 min each time and fixed with 4 % paraformaldehyde at 4 °C for 4 h. Then, they were washed again for three times with PBS for 5 min each time and blocked by incubating with 5 % BSA in 0.1 % Triton 9 100 for 30 min at 37 °C for 30 min. Then, they were washed with PBS for 3 times (5 min each time), and incubated overnight at 4 °C with primary antibody, the anti-rat Thy1.1 mAb, 1:200 dilution, and anti-rat Map2 pAb, 1:400 dilution. After the incubation, the cells are washed with PBS for 3 times (5 min each time), and incubated with secondary antibodies at 37 °C for 1 h (TRITC-labled goat anti-mouse IgG and FITC-labeled goat anti-rabbit IgG). The cells are again washed with PBS (5 min each time), mounted on the slides using buffered glycerin and observed under fluorescent microscope, and photographs of general and fluorescent images were taken of the same field. The ratio of double positive cells to total cells was considered as the degree of RGCs purity. Five fields were chosen each time, and a total of 10 fields were used to calculate the purity of cells.

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Fluorochrome dye JC-1 was used to evaluate the changes in mitochondrial membrane potential. Cells were seeded in poly-L-lysine-coated 6-well plates at a density of 5 9 105/ well and when 80 % confluent, the cells were treated with glucose and EPO for 48 h, and then stained with JC-1 according to the manufacturer’s instructions. Cells were then harvested, gently rinsed with PBS, and resuspended in 500 ll PBS. Fluorescent intensity of the cells was quantitatively analyzed by flow cytometry. Measurement of ROS, Lipid Peroxidation, Antioxidation Enzymes, and Total Antioxidative Capacity (T-AOC) Cells were seeded at a density of 5 9 104/well of a 6-well plate, and allowed to grow until 80 % confluent. Then, they were grown for further 48 h as untreated, with EPO alone, with 30 mM glucose without or with various concentrations of EPO, 10 U, 20 U, 40 U, and NAC (5 mmol/L). After washing with PBS, 10 lmol/L of DCFH-DA (1 ml) was added, mixed, and incubated for 20 min at 37 °C in dark according to the instructions from the ROS detection

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kit manufacturer. Then, the cells were washed with serumfree culture medium for three times and analyzed by flow cytometry. The SOD, GSH-Px, CAT, T-AOC, and malonaldehyde (MDA) were measured following the protocol. The cells treated with NAC were examined only for the MDA content.

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Keuls was used as a post-hoc test. p values of less than 0.05 were considered to represent statistical significance.

Results RGCs Identification

Western Blotting Cell lysates corresponding to 40 mg protein were loaded on SDS-PAGE gel and electrophoresed. Proteins separated on the gel were electroblotted onto a PVDF membrane. The membranes were blocked with 5 % non-fat milk in TBST (20 mM Tris, 150 mM NaCl, pH 7.5 and 0.1 % Tween 20) and then incubated overnight at 4 °C with primary antibodies for cytochrome c (1:600), anti-Bax (1:400), anti-Bcl-2 (1:400), caspase-3 (1:400), caspase-9 (1:400), and b-actin (1:700). Then, the membranes were incubated with secondary antibodies (Peroxidase-Conjugated AffiniPure Goat AntiRabbit lgG, 1:5,000). Excess antibody was washed off with 20 mM TBST, and the proteins of interest were developed with ECL and exposed to the imaging film. The bands were scanned to measure the densities using an automatic image analysis system (Alpha Innotech Corporation, San Leandro, CA, USA), and they were normalized to b-actin. Statistical Analysis Data are expressed as mean ± SD. Statistical comparisons were made using one-way ANOVA. Student–Newman–

At day 5 of culturing, the cells were labeled with Thy1.1 mAb and Map2 pAb (shown in Fig. 1) to identify the cells. Effect of EPO on High Glucose-Induced Cell Apoptosis in RGCs The results of Hoechst33358 staining (Fig. 2) show that high glucose treatment of the RGCs caused a significant increase in their apoptosis compared to the control (P \ 0.01). The cells grown in the presence of different concentrations of EPO showed a significant attenuation of high glucose-induced apoptosis (P \ 0.01), indicating the protective effect of EPO. Effect of EPO on ROS, MDA, and SOD Levels in the RGCs Treated with High Glucose The RGCs treated with high concentrations of glucose exhibited significantly higher levels of ROS and malonaldehyde (MDA) which were attenuated with EPO (P \ 0.01, Fig. 3a, b). The levels of SOD, GSH-PX, CAT,

Fig. 1 Imaging of the double-labeled RGCs by phase contrast microscopy at 100 9 magnification, a gray image, b red fluorescence image representing Thy1.1 labeling, c green image representing Map2 labeling, d superimposed red and green fluorescent images (Color figure online)

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significantly decreased compared to the controls (P \ 0.01). However, the EPO pretreatment significantly blocked this effect of glucose (P \ 0.05) (Fig. 4). Effect of EPO on Cytochrome C Release

Fig. 2 Effect of EPO on High Glucose-induced apoptosis in RGCs. Asterisk and hash represent significant effect compared to the glucose (P \ 0.001) exposed or control cells (P \ 0.01), respectively

and T-AOC decreased with high glucose were significantly recovered by EPO pretreatment (P \ 0.05 or P \ 0.01) (Fig. 3c, d, e, f). EPO Restored the Damage to Mitochondrial Membrane in RGCs Treated with High Glucose The JCI-produced red florescence intensity, the measure of Dwm, of the cells treated with high glucose was Fig. 3 Effect of EPO on levels of ROS (a), LPO (b), SOD (c), Gpx (d), CAT (e), and T-AOC (f) in RGCs exposed to high concentrations of glucose. Hash and asterisk represent significant effect compared to untreated (P \ 0.001) and high glucose-exposed (*P \ 0.05 or **P \ 0.01) cells, respectively

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The treatment of RGCs with glucose led to a significantly elevated (P \ 0.001) content of cytochrome C in the cytosol accompanied with a substantially reduced (P \ 0.001) expression of this protein in the mitochondria (Fig. 5). This effect was restored with EPO dose dependently indicating the crucial role of cytochrome C release from the mitochondria in high glucose-induced apoptosis. Effect of EPO on the Bax and BCl-2 Expressions in RGCs Exposed to High Glucose As shown by the Western blots in Fig. 6, the expression of pro-apoptotic protein, Bax, was significantly up-regulated in high glucose-treated RGCs compared to the untreated cells (P \ 0.01). In contrast, in the cells pretreated with EPO, the high glucose-mediated increase in Bax expression was significantly and dose dependently reduced (P \ 0.01). The expression of anti-apoptotic Bcl-2 remained unaltered in the presence or absence of EPO.

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Fig. 4 Effect of EPO on change in mitochondrial membrane potential (Dwm) induced with high concentration of glucose in RGCs. Asterisk and hash represent significant effect compared to control (P \ 0.01) and glucose-exposed (P \ 0.05) cells, respectively

Fig. 6 Effect of EPO treatment on the ratio of Bax and Bcl-2 expressions (Bax/Bcl-2) in high glucose-treated RGCs. Asterisk and hash represent significant effect compared to untreated (P \ 0.01) and high glucose-treated (P \ 0.01) cells, respectively

Discussion

Fig. 5 Effect of EPO on high glucose-induced release of mitochondrial cytochrome C in RGCs determined by Western blotting. Asterisk and hash represent significant effect compared to control (P \ 0.001) and glucose-treated (P \ 0.05) cells, respectively

Effect of EPO on the Expressions of Caspase-3 and Caspase-9 in RGCs Treated with High Glucose As shown in Fig. 7, compared to the untreated, the high glucose-exposed cells exhibited significantly increased expressions of caspase-3 and caspase-9 (P \ 0.01). In contrast, the cells pretreated with different concentrations of EPO or NAC showed a restoration of the high glucose-induced increase in caspase-3 and caspase-9 expressions (P \ 0.01), indicating the preventive effect of EPO.

DR, a common complication of DM, is frequently known to cause blindness. Kern et al. [11] found that among the neuronal cells, RGCs were the first to be affected by DR and they apoptized with a high rate. EPO, a glycoprotein with a molecular weight of 34 kDa, has been found to reduce apoptosis during brain development and provides neuroprotection against hypoxia in murine embryonic cells [12]. It has also been reported to exert neuroprotection against experimental brain injuries and inflammation caused by ischemia or concussion [13–15]. Most importantly, the protective effect of EPO has been observed against the retinal neuron injuries caused by various pathophysiological factors including acute ischemia–reperfusion and TNF-a [16–21]. The antioxidant action of EPO is mediated through enhancing the activity of SOD, GSH-Px, and CAT [22–24]. In this study, we established high glucose-mediated apoptosis in RGCs, which was confirmed by Hoechest 33358 staining. We found that EPO exerted a dosedependent decrease in high glucose-mediated elevation of the apoptotic rate. Accumulating evidence has shown that excessive ROS is generated as a result of the exposure of cells to high concentrations of glucose and adds to the complications of DM [25]. In this study, we found that EPO treatment attenuated the glucose-induced increase in ROS and MDA levels, and significantly elevated T-AOC, and up-regulated the levels of SOD, GSH-PX, and CAT (P \ 0.05 or P \ 0.01).

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Fig. 7 Effect of EPO treatment on the expressions of caspase-3 and caspase-9 in high glucosetreated RGCs. Asterisk and hash represent significant effect compared to the untreated (P \ 0.01) and high glucosetreated (P \ 0.01) cells, respectively

Mitochondrial DNA and membrane are particularly sensitive to oxidative damage caused by ROS. The reduction in mitochondrial membrane potential causes its depolarization that leads to the opening of permeable transition pore (mtPTP), thus allowing an outflow of Cyt c, a process that is indicative of early apoptosis [6]. Our results demonstrated an enhancement of apoptosis and decrease in mitochondrial membrane potential in RGCs treated with high glucose indicating that the two phenomena were correlated. The dose-dependent inhibition of the deleterious effects of high glucose on membrane potential and concomitant recovery from apoptosis suggests the effectiveness of EPO in restoring the damage. The membrane damage is also associated with decreased ATP synthesis, imbalance of intracellular calcium, release of apoptosis inducing factor (AIF), caspase-9, and cytochrome C [26–28]. The cyt C released as a consequence of increased membrane permeability is known to trigger the activation of caspases, the pro-apoptotic enzymes. As demonstrated by our results, EPO effectively inhibited the high glucose-mediated up-regulation of caspase-9 and caspase-3 in a dose-dependent manner. In addition, high glucose exposure of the cells resulted in increased Bax/Bcl2 ratio, which is an indication of elevated apoptotic and suppressed anti-apoptosic activities. We observed that EPO produced a dose-dependent restoration of the Bax expression and decrease in Bax/Bcl-2 ratio. Treatment of the high glucose-exposed cells with EPO also caused a down-regulation of the overly expressed caspase-3 and caspase-9 dose dependently. Thus, our results suggest that EPO ameliorates the degeneration of RGCs by inhibiting the generation of ROS, enhancing the total antioxidant status of the cells through the up-regulation of SOD, GSH-PX, and CAT. In the absence of exaggerated ROS signal, the downstream cascade of events including increase in lipid peroxidation, mitochondrial membrane permeability, release of Cyt c, up-regulation of pro-apoptotic proteins, Bax and caspases, the apoptosis, and subsequent cellular degeneration was

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prevented. These observations suggest the therapeutic potential of EPO for the management of diabetes-related RGC degeneration.

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Erythropoietin (EPO) protects against high glucose-induced apoptosis in retinal ganglional cells.

The aim of this study was to investigate the protective effect and mechanism of EPO on the apoptosis induced by high levels of glucose in retinal gang...
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