Cell Mol Neurobiol DOI 10.1007/s10571-015-0231-5
ORIGINAL RESEARCH
Neuroprotective Effect of Lutein on NMDA-Induced Retinal Ganglion Cell Injury in Rat Retina Chanjuan Zhang1 • Zhen Wang1 • Jiayi Zhao1 • Qin Li1 • Cuiqin Huang1 Lihong Zhu1 • Daxiang Lu1
•
Received: 18 April 2015 / Accepted: 20 June 2015 Ó Springer Science+Business Media New York 2015
Abstract Lutein injection is a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate its protective effects in retinal ganglion cells (RGCs) against N-methyl-D-aspartate (NMDA)-induced retinal damage in vivo. Retinal damage was induced by intravitreal NMDA injection in rats. Each animal was given five daily intraperitoneal injections of Lutein or vehicle along with intravitreal NMDA injections. Electroretinograms were recorded. The number of viable RGCs was quantified using the retinal whole-mount method by immunofluorescence. Proteins were measured by Western blot assays. Lutein reduced the retinal damage and improved the response to light, as shown by an animal behavior assay (the black-and-white box method) in rats. Furthermore, Lutein treatment prevented the NMDA-induced reduction in phNR wave amplitude. Lutein increased RGC number after NMDAinduced retina damage. Most importantly, Bax, cytochrome c, p-p38 MAPK, and p–c-Jun were all upregulated in rats injected with NMDA, but these expression patterns were reversed by continuous Lutein uptake. Bcl-2, p-GSK-3b, and p-Akt in the Lutein-treated eyes were increased compared with the NMDA group. Lutein has neuroprotective effects against retinal damage, its protective effects may be partly mediated by its anti-excitability neurotoxicity, through MAPKs and PI3K/Akt signaling, suggesting a potential approach for suppressing retinal neural damage.
& Daxiang Lu [email protected] 1
Key Laboratory of State Administration of Traditional Chinese Medicine of China, Department of Pathophysiology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong Province, China
Keywords Lutein Apoptosis MAPKs signaling PI3K/ Akt signaling Retinal ganglion cell Abbreviations RGCs Retinal ganglion cells ERG Electroretinograms phNR Photic negative response RIPA Radio immunoprecipitation assay SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis PVDF Polyvinylidene fluoride ECL Electrochemiluminescence AkT Protein kinase B/PKB ERK Extracellular signal-regulated kinase GSK-3b Glycogen synthase kinase-3b MAPK Mitogen-activated protein kinases
Introduction Lutein is a xanthophyll dietary carotenoid that is structurally similar to zeaxanthin (Sies et al. 1992). Lutein has a chemical formula of C40H56O2 with a long carbon chain with alternating single and double carbon–carbon bonds with attached methyl side groups. At both ends of the carbon backbone the molecule contains a cyclohexene structure with an attached hydroxyl group (Granado et al. 2003). The presence of a hydroxyl group at both ends of the molecule distinguishes Lutein from other carotenoids (Kijlstra et al. 2012). The characteristic structure enables Lutein to react more strongly with singlet oxygen than other carotenoids (Tian et al. 2007). Lutein is predominately present in the macular region and acts as an efficient pigment for absorbing high-energy
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blue light. It is also a direct free radical scavenger to prevent macular damage (Johnson 2014). It is anti-apoptotic and plays a neuroprotective role by decreasing oxidative stress (Wang et al. 2013). However, the effect of Lutein on specific cell populations, such as retinal ganglion cells (RGCs), is unknown. Lutein possesses biological antioxidant activity, and it is a major component of carotenoids with non-provitamin A activity (Wang and Lin 2008). We sought to investigate whether Lutein could reverse the excitotoxicity in RGCs. It can inhibit the increased PI3K activity and Akt phosphorylation after oxidative stress (Lee et al. 2006), which provides a basis for us to investigate the role of Lutein in RGC protection. RGCs are neurons that transmit visual signals from the retina to the brain and are the sole output neurons of the retina. RGCs are the main cells expressing N-methyl-D-aspartate receptors (NMDARs) in retinal neurons, which render these cells susceptible to glutamate excitotoxicity (Huang et al. 2014). RGC dysfunction or loss is associated with various optic neuropathies, including glaucoma (Zhang et al. 2014), diabetic retinopathy (Yuan et al. 2014), optic neuritis (Suhs et al. 2014), or Leber’s hereditary optic neuropathy (Gallenmuller and Klopstock 2014). RGC death may occur via a variety of mechanisms involving, for example, reactive oxygen species (Li et al. 2009), excitatory amino acids (Dun et al. 2007), nitric oxide (Rathnasamy et al. 2014), or apoptosis (Dvoriantchikova et al. 2014). Because of RGC susceptibility to different types of damage and because of their importance, it is extremely urgent to find strategies to protect these cells. Neuroprotective therapies that focus on factors leading to RGC degeneration have attracted increasing attention. Studies on the protective effects of carotenoids (including Lutein) against retinal damage have primarily focused on age-related maculopathy (Parisi et al. 2008), but it is necessary to investigate methods to protect RGCs from all mechanisms of damage. According to its properties and function, we believe that Lutein may be the basis for such a therapeutic strategy. The purpose of this study was to examine the effects and possible mechanisms of Lutein on retinal damage in vivo. We studied the effects of Lutein against NMDA-induced retinal damage in rats.
Materials and Methods Animals Female Sprague–Dawley (SD) rats (body weight 200–250 g) were kept under 12-h light/12-h dark conditions. All animal procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National
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Institutes of Health. And we make all efforts to minimize the suffering and number of animals used. Rats were randomly divided into six experimental groups and each group containing 15 rats: a control group, a sham group, NMDA group, NMDA and Lutein (low concentration, middle concentration, and high concentration) group. NMDA-Induced Retinal Damage Model and Treatment with Lutein Retinal damage was induced by N-methyl-D-aspartic acid (NMDA, Sigma, St. Louis, MO, USA) as described by Siliprandi et al. (1992). Briefly, the animals were anesthetized with 10 % chloral hydrate (0.38 ml/100 g; 302170, Kermel, Tian Jing, China) by intraperitoneal injection. Body temperature was maintained at between 37.0 and 37.5 °C with the aid of a heating pad and heating lamp. Retinal damage was induced by the injection (1 ll/ eye) of NMDA dissolved at 40 mM in 0.9 % normal saline (NS), which was injected into the vitreous body of the eyes under anesthesia. One drop of 0.01 % chloramphenicol ophthalmic solution (Yuan Da Pharmaceuticals, Wu Han, China) was applied topically to the treated eye immediately after the intravitreal injection. On the fourth day after the NMDA injection, the rats were employed for visual behavior detection and the next day they were allocated for ERG recording, then the eyeballs were enucleated for immunofluorescence, histological, and Western blot analyses. Lutein (0.25, 0.5, or 1.0 mg/kg,) was dissolved in corn oil immediately before use and intraperitoneally injected five times (0, 1, 2, 3, and 4 days after the NMDA injection). Visual Behavior Detection The visual function of the retina could be evaluated by a black-and-white box, which is formed by two chambers (a white and a black chamber) with a door opening between them (Fig. 1). This system was employed to test the white– black visual discrimination of the rats (Lin et al. 2008). An aperture (10 9 12 cm) between the black-and-white chambers allowed the rats to travel freely from one to the other. The black chamber was illuminated with an infrared light, while the white chamber was illuminated by a bright white light. Two cameras, installed in the two chambers separately, captured the activities of the rats and were connected to a Noldus EthoVision XT 8.0 recorder and monitor. The rats were placed into the middle of the white chamber at the start of the trial and left in the box for 5 min. The number of intercompartmental crosses, the distance moved, the mean velocity, and the time spent in each chamber were recorded by the Noldus EthoVision XT 8.0 software.
Cell Mol Neurobiol Fig. 1 Behavioral assay of rats in the black-and-white box. The red lines represent the moving trajectory of a single rat in the two experimental groups. The representative rats in the control group spent more time traveling in the black chamber than in the white chamber (a), as did the Sham group (b). In the NMDA group, the rats spent almost equal time in the black chamber and in the white chamber (c). Lutein increased the time the animals spent in the black chamber (d, e, f). 0.25L ? N = 0.25 mg/kg Lutein ? NMDA group; 0.5L ? N = 0.5 mg/kg Lutein ? NMDA group; 1.0L ? N = 1.0 mg/kg Lutein ? NMDA group. g, h The statistical graphs of the rats’ time in the black chamber and white chamber. The results are reported as the mean ± SEM. *P \ 0.05 versus the control group, # P \ 0.01 versus the NMDA group, n = 15 (Color figure online)
Electroretinography (ERG) Functional signals and activities of each cell type could be recorded by electroretinography (ERG), and the photopic negative response (PhNR, induced by RGCs) (Chen et al. 2008) was analyzed to determine the change in RGCs after intravitreal injection of NMDA. Rats should be adapted to the dark before ERG recordings to reach a stable status. The time for dark adaptation in the present experiment was at least 70 min (Wang et al. 2011). After stabilization, the ERGs were recorded. In brief, the control rats were anesthetized with 10 % chloral hydrate (0.38 ml/100 g), and the pupils were dilated with tropicamide. Recording electrodes were placed on the corneas. Two reference electrodes were inserted into subcutaneous tissue before the ears, and one ground electrode was inserted into the subcutaneous tissue of the tail, as in a previous study (Zhao et al. 2013). The a-wave, b-wave, and PhNR were recorded using a Roland Consult electrophysiological diagnostic system (Brandenburg, Germany). Scotopic ERGs (a-wave, b-wave; 3.0 and 10.0 cds/m2, white flash) were first
recorded after an 8-h dark adaptation. After light adaption under a continuous blue background (25 cds/m2) for 5 min to suppress rod cell electrical activity, PhNR was recorded with red flashes (3.0 and 10.0 cds/m2). The amplitude of the a-wave was measured from its baseline to its peak, and the amplitude of the b-wave was measured from the trough of the a-wave to the peak of the b-wave. The amplitude of PhNR was defined as the first negative peak following the b-wave that was measured relative to the baseline. PhNR wave was measured using pCLAMP_10.2 software after applying 50 Hz low-pass filtering. Retina Immunofluorescence and the Retinal Whole Mounts The rats were anesthetized by an intraperitoneal injection of 10 % chloral hydrate on the fifth day after intravitreal injection of NMDA (40 mM) at 1 ll/eye. Each eye was enucleated; then, the retina was moved off the eye cup and immersed for 30 min at room temperature in a fixative solution containing 4 % paraformaldehyde dissolved by
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PBS (pH 7.4). After washing six times with PBS, the retina was blocked in 5 % donkey serum in Tris–phosphate buffer with 3 % Triton 100 (PBST) for 1 h at room temperature. Then, the retina was incubated with a primary antibody goat polyclonal antibody against Brn-3 (1:100, Santa Cruz Biotechnology) for 48 h at 4 °C after that the primary antibody was removed and the retina was rinsed six times with PBST (containing 0.3 % Triton 100), then it was incubated with appropriate fluorescent secondary antibody dylight 488-donkey anti-goat IgG (1:1000, EarthOx) for 3 h at room temperature and then the secondary antibody was removed and the retina was rinsed six times with PBST (containing 0.3 % Triton 100) again, at last the antiquenching solution was added to the retina for capturing images using a Leica epifluorescence microscope to count the whole-mounted RGCs with the 1.5 mm interval lattice method, then every lattice field is photographed, after that the RGCs are numbered, then the average number in one lattice field is calculated, at last the density of the RGCs in the whole retina is calculated to make the statistical graph. Western Blot Analysis The rats were anesthetized by an intraperitoneal injection of 10 % chloral hydrate on the fifth day after intravitreal injection of NMDA (40 mM) at 1 ll/eye. The eye was enucleated and the retina was detached, then lysed in icecold RIPA lysis buffer (Beyotime, Nantong, Jiangsu, China) and for full cleavage the retina lysate was broken using the ultrasonic instrument. The lysate was centrifuged for 15 min at 12,000 rpm, 4 °C, and the supernatants were collected for protein analysis. The total protein concentration was determined using an Enhanced BCA Protein assay kit (Beyotime, Nantong, Jiangsu, China). Equal amounts of proteins (40 lg) were then separated by 12 % SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking in Tris–phosphate buffer with 0.1 % Tween 20 (TBST) containing 5 % non-fat milk and washing with TBST, the membranes were incubated with primary antibodies obtained from Cell Signaling Technology (rabbit, 1:1000) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated anti-rabbit antibody (1:5000, Cell Signaling Technology, Inc. Danvers, MA, USA) at room temperature for 2 h. Signals were detected on a gel imaging system using the ECL Western blotting substrate (Millipore, Billerica, MA, USA). Statistical Analysis Statistical analysis was performed with SPSS software, version 19.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) followed by the least
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significant difference multiple comparison test was used. P \ 0.05 was considered significant.
Results Alteration of Visual Behavioral After Intravitreal Injection NMDA in SD Rats Rats prefer dark and narrow places. Therefore, in the blackand-white box test, when a rat is placed in the center of a white chamber, it will enter the black chamber through an aperture between the white and black chamber. Rats stayed in the dark chamber for a longer time than in the white chamber in both the control groups (black duration 229.7 ± 6.7 s; white duration 70.2 ± 6.7 s, Fig. 1a) and the sham group (black duration 220.5 ± 5.8 s; white duration 79.5 ± 6.0 s, Fig. 1b), while the rats spent similar times in the dark and white chambers in the NMDA group (black duration 162.7 ± 6.8 s; white duration 137.3 ± 6.8 s, Fig. 1c). However, the time rats stayed in the dark was extended by Lutein treatment (Fig. 1d–f). There was a significant difference in the duration in the dark chamber between the control group (229.7 ± 6.7 s) and NMDA group (162.7 ± 6.8 s, P \ 0.01, Fig. 1a, c). Compared with the NMDA group, the duration in the dark chamber was extended in the 0.25-mg/kg Lutein group (black duration 187.9 ± 10.0 s; white duration 112.1 ± 10.0 s), the 0.5-mg/ kg Lutein group (black duration 216.3 ± 7.3 s; white duration 83.7 ± 7.3 s), and the 1.0-mg/kg Lutein group (black duration 217 ± 11.3 s; white duration 82.9 ± 11.3 s). Significant differences were observed among the NMDA group, the 0.5-mg/kg Lutein group (216.3 ± 7.3 s), and the 1.0-mg/kg Lutein group (217 ± 11.3 s, P \ 0.01). Different PhNR Amplitude Changes in ERG Recordings After Intravitreal Injection of NMDA PhNR is a negative-going wave that occurs following the b-wave. Because its origin remains undetermined, PhNR is significantly reduced in human patients with primary openangle glaucoma (Huang et al. 2012), anterior ischemic optic neuropathy (Rangaswamy et al. 2004), and other optic nerve neuropathies (Machida et al. 2004), consistent with an origin in ganglion cells or their axons. There was no significant difference in PhNR amplitude between the control group (14.3 ± 1.6 lV) and the sham group (13.3 ± 0.9 lV), but there was a significant difference between the control group (14.3 ± 1.6 lV) and the NMDA group (4.8 ± 0.7 lV; P \ 0.01). Compared with the NMDA group (4.8 ± 0.7 lV), the PhNR amplitude increased in the 0.25-mg/kg Lutein group, in the 0.5-mg/kg
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Lutein group, and in the 1.0-mg/kg Lutein group. A significant difference was observed between the NMDA group (4.8 ± 0.7 lV) and the 0.5-mg/kg Lutein group (12.4 ± 1.8 lV; P \ 0.01), but there were no significant differences among the NMDA group, the 0.25-mg/kg Lutein group (8.0 ± 0.8 lV), and the 1.0-mg/kg Lutein group (7.5 ± 1.3 lV; Fig. 2b). The Change in RGC Number After Intravitreal Injection NMDA by Retinal Whole Mounts On the fifth day, rat eyes were enucleated after sacrifice; then, the retinas were separated from the cup of the eyes and exposed to a primary antibody against Brn-3 to count the RGCs. There was no significant difference in RGC number between the control group (884 ± 30 cells/mm2; Fig. 3a) and the sham group (788 ± 23 cells/mm2; Fig. 3b), but there was a significant difference between the control group (884 ± 30 cells/mm2) and the NMDA group (101 ± 7 cells/mm2; Fig. 3c). Compared with the NMDA group, the number of RGCs was increased in the 0.25-mg/ kg Lutein group (177 ± 13 cells/mm2; Fig. 3d), in the 0.5mg/kg Lutein group (256 ± 16 cells/mm2; Fig. 3e), and in the 1.0-mg/kg Lutein group (162 ± 17 cells/mm2; Fig. 3f). And significant differences among the 0.5-mg/kg Lutein group (256 ± 16 cells/mm2) and the NMDA group (101 ± 7 cells/mm2). Effects of Lutein on Expression of Bax, Bcl-2, Caspase-3, and Cytochrome c in NMDA-Induced Retinas To study the role of Lutein in NMDA-induced retina injury, we analyzed the levels of apoptosis markers in the retinas using Western blotting. Cleaved caspase-3, the
activated form of caspase-3, was clearly upregulated in the NMDA group compared with the control group. The same was true of the pro-apoptotic proteins Bax and cytochrome c (P \ 0.05; Fig. 4). In contrast, the anti-apoptotic protein Bcl-2 was clearly increased in the 0.5-mg/kg Lutein group, and the ratio of Bax to Bcl-2 significantly declined in the 0.5-mg/kg Lutein group compared with the NMDA group (P \ 0.05; Fig. 4). Effect of Lutein on Expression of ERK, p38 MAPK, and c-Jun in NMDA-Induced Retina Damage ERK, p38 MAPK, and c-Jun were also analyzed by Western blot. Phosphorylated p38 MAPK and c-Jun were significantly elevated in the NMDA group compared with the control group (P \ 0.05). These increases were inhibited by constant Lutein intake, especially 0.5 mg/kg Lutein, compared with the NMDA group (P \ 0.05; Fig. 5). Phosphorylated ERK had almost no change in all the groups. Effect of Lutein on Expression of Akt and GSK-3b in NMDA-Induced Retinas To clarify the effect of Lutein on Akt activation in NMDAinduced retina damage, we performed immunoblot analyses of phosphorylated (activated) Akt. The activation of Akt in the retina was significantly increased by Lutein intake, particularly the 0.5-mg/kg dose (Fig. 6), compared with the NMDA group (P \ 0.05). GSK-3b, a downstream protein of Akt, was significantly increased in the 0.5-mg/kg Lutein group (Fig. 6) compared with the NMDA group (P \ 0.05). There were no significant difference in total Akt or total GSK-3b between groups (Fig. 6; n = 5).
Fig. 2 The effects of Lutein on NMDA-induced changes in ERG in SD rats. a The normal ERG. There was an obvious PhNR wave. b Statistical analysis of the mean amplitudes of the PhNR waves of photopia 3.0. The PhNR wave amplitude of ERG was significantly lower in the NMDA group, but the effects induced by NMDA were largely reversed by Lutein (0.5 mg/kg). The data in all bar graphs are shown relative to controls. The data represent the mean ± SEM. *P \ 0.05 versus control; #P \ 0.01 versus the NMDA group, n = 13
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Cell Mol Neurobiol Fig. 3 The effects of Lutein on NMDA-induced change in the number of RGCs in SD rats. The number of RGCs in the NMDA group (c) was significantly reduced compared with control (a) using Brn-3-marked cells. Lutein (d, e, f) increased the number of Brn-3-positive cells in retina, especially 0.5 mg/kg Lutein (e). 0.25L ? N = 0.25 mg/kg Lutein ? NMDA group; 0.5L ? N = 0.5 mg/kg Lutein ? NMDA group; 1.0L ? N = 1.0 mg/kg Lutein ? NMDA group. (g) Statistical analysis of the mean number of RGCs. The number of RGCs was significantly reduced in the NMDA group, but the effects induced by NMDA were largely reversed by Lutein (0.5 mg/kg). The data in all bar graphs are shown relative to controls. The data represent the mean ± SEM. *P \ 0.05 versus control; #P \ 0.01 versus NMDA group, n = 9
Discussion Excitability neurotoxicity was common cause of many ocular diseases, which lead to irreversible RGC damage and RGCs are sensitive to excitability neurotoxicity in pathological situations in vivo and in vitro (Yamasaki et al. 2005). In pathologies such as ischemia/reperfusion, glaucoma, and DR, the overproduction of ROS, free radicals, and excitatory amino acids overwhelm the intrinsic eliminating mechanism (Osborne et al. 2004; Tezel 2006), and these events exceed the ability of the normal retina to protect against excitability neurotoxicity which leads to RGC damage. NMDA-induced apoptosis in RGCs is caspase independent (Kim et al. 2013) and yet includes the Akt/PI3K, the p38 MAPK, and the ERK signaling
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pathways in previous study (Nakazawa et al. 2005; Manabe and Lipton 2003; Lin et al. 2007). In our experiment we investigated the influence of Lutein on NMDA-induced retina excitability neurotoxicity using a combination of the retina whole-cell mount, immunohistochemistry, ERG, visual behavior detection, and Western blotting. We found significant improvement in the RGCs treated with Lutein compared with the injury group, indicating that Lutein improved the ability of RGCs to avoid excitability neurotoxicity. Lutein blocks excitability neurotoxicity, oxidative stress, inflammation, and apoptosis, and the agent is neuroprotective against the NMDA-induced injury (Miyake et al. 2014; Sun et al. 2014; Woo et al. 2013; Li et al. 2009, 2012a, b). The mechanism of Lutein’s regulation of cell damage and
Cell Mol Neurobiol Fig. 4 The expression of apoptotic proteins Bax, Bcl-2, caspase-3, and cytochrome c in NMDA-induced retina injury by Western blot. a The Bax and Bcl-2 protein expression in NMDA-induced retina. The ratio of Bax/Bcl-2 was significantly increased in the NMDA group compared with the control group, but this was reversed by Lutein uptake. b The caspase-3 protein expression in NMDA-induced retina. The ratio of cleaved caspase-3/caspase-3 was significantly elevated in the NMDA group compared with the control group, but this was also reversed by Lutein uptake. c The cytochrome c protein expression in NMDA-induced retina. The expression of cytochrome c was significantly increased in the NMDA group compared with the control group, but this was again reversed by Lutein uptake. The results are reported as the mean ± SEM. #P \ 0.01 versus the control group, *P \ 0.01 versus the NMDA group (n = 6)
apoptosis is through the members of the Bcl-2 family, the MAPK pathway (Lo et al. 2012, 2013), and the PI3K/Akt pathway. The Bcl-2 family consists of both apoptosis promoters, including Bax and Bad, and apoptosis inhibitors, which include Bcl-2, Bcl-X, and Mcl-1. A family of aspartate-specific proteases termed caspases is activated in the execution phase of apoptosis. The cleavage of caspase-3 is often viewed as the final step of the process that promotes the end of the apoptotic signaling pathway. In addition, the ratio of Bax/Bcl-2 might determine survival or death (Verma et al. 2013). In vivo, we found that Lutein improved the response to light of NMDA-damaged rats in the black-andwhite box assay (Fig. 1), increased the PhNR wave amplitude of NMDA-damaged rats according to ERG recordings (Fig. 2), and rescued the RGC decrease induced by NMDA as shown by whole-cell mount technology (Fig. 3). Then, we found that the expression of Bax, cleaved caspase-3, and cytochrome c was inhibited by Lutein, while the expression of Bcl-2 was promoted by Lutein. The middle dose (0.5 m/ kg) of Lutein had the optimal effect (Fig. 4), which was in
agreement with a previous finding (Han et al. 2012) and (Chen et al. 2012). The MAPKs is a kind of serine or threonine protein kinase, a major signaling pathway in cellular stress and damage response, and the MAPK signaling pathway is believed to regulate the apoptosis of retina cells after ischemia/reperfusion damage (Ishizuka et al. 2013). The kinase family has three members in the classical pathway, including extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and the protein kinase p38 (Xu et al. 2014). ROS-activated ERK contribute to synaptophysin degradation and neuronal dysfunction in the diabetic retina (Sasaki et al. 2010), while JNK activation has the opposite effect. The effect of p38 on apoptosis remains controversial (Ishizuka et al. 2013; Li et al. 2012). Our data agree with these. In the present investigation, we found that the activation of p38 MAPK and c-Jun were suppressed by Lutein, indicating that Lutein protects against NMDA-induced RGC damage through the MAPK pathway (Fig. 5).
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Fig. 5 Western blots of phosphorylated ERK, p38 MAPK, and c-Jun in NMDA-induced retina injury. a Phosphorylated ERK in NMDAinduced retina. b Phosphorylated p38 MAPK was higher in the NMDA group compared with the control group, but Lutein reversed
this. c Phosphorylated c-Jun was higher in the NMDA group compared with the control group, but Lutein reversed this. The results are reported as mean ± SEM. #P \ 0.01 versus the control group, *P \ 0.01 versus the NMDA group (n = 5)
The serine/threonine kinase Akt, also named protein kinase B (PKB), plays a critical role in many cellular activities, including cell survival, growth, proliferation, angiogenesis, metabolism, and migration (Manning and Cantley 2007). Phosphorylated Akt has a neuroprotective effect in a diabetic rat model (Park et al. 2014). We also found that Lutein increased Akt activation in NMDA-induced retina damage, and p-Akt activated its downstream protein GSK-3b to carry out its neuroprotection function (Fig. 6). Taken as a whole, our data demonstrate that Lutein exerts anti-apoptotic effects during NMDA-induced RGC injury by activating Akt and inhibiting the phosphorylation of p38 MAPK and c-Jun, which result in increased Bcl-2 levels and reduced levels of Bax, caspase-3, and
cytochrome c. Thus, our results provide important insights into the potential mechanisms involved in the neuroprotective effects of Lutein, which may make it a candidate for a neuroprotective agent against ocular damage. However, we found that the neuroprotective effects of Lutein were not completely in a dose-dependent manner during NMDA-induced RGC injury, and the middle concentration of Lutein (0.5 mg/kg) played a better effect, which may be due to the high concentration of lutein in vivo conversion of other carotenoids, then competed with Lutein to bind to lutein-binding protein, resulting in the decreased retinal Lutein content. Research shows that the increasing of Lutein in retina could reduce light-induced photoreceptor apoptosis while the increasing of Lutein in the serum does not have the effect (Thomson et al. 2002). Further studies
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Cell Mol Neurobiol Fig. 6 Western blots of phosphorylated Akt and GSK3b in NMDA-induced retina damage. a The p-Akt/Akt ratio in NMDA-induced retina was increased, but this was reversed in the Lutein group. b The p-GSK-3b/GSK-3b ratio was significantly increased in the NMDA group, but this was also reversed in the Lutein group. The results are reported as the mean ± SEM. #P \ 0.01 versus the control group, *P \ 0.01 versus NMDA group (n = 5)
are needed to elucidate the exact mechanism by which Lutein protects RGCs. Acknowledgments This work was supported by grants from the National Program on Key Basic Research Project (973 Program No. 2011CB707501), and the National Natural Science Foundation of China (Nos. 81371442 and 81471236). Compliance with Ethical Standards Conflict of interest No conflicting relationship exists for any author.
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ORIGINAL RESEARCH
Neuroprotective Effect of Lutein on NMDA-Induced Retinal Ganglion Cell Injury in Rat Retina Chanjuan Zhang1 • Zhen Wang1 • Jiayi Zhao1 • Qin Li1 • Cuiqin Huang1 Lihong Zhu1 • Daxiang Lu1
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Received: 18 April 2015 / Accepted: 20 June 2015 Ó Springer Science+Business Media New York 2015
Abstract Lutein injection is a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate its protective effects in retinal ganglion cells (RGCs) against N-methyl-D-aspartate (NMDA)-induced retinal damage in vivo. Retinal damage was induced by intravitreal NMDA injection in rats. Each animal was given five daily intraperitoneal injections of Lutein or vehicle along with intravitreal NMDA injections. Electroretinograms were recorded. The number of viable RGCs was quantified using the retinal whole-mount method by immunofluorescence. Proteins were measured by Western blot assays. Lutein reduced the retinal damage and improved the response to light, as shown by an animal behavior assay (the black-and-white box method) in rats. Furthermore, Lutein treatment prevented the NMDA-induced reduction in phNR wave amplitude. Lutein increased RGC number after NMDAinduced retina damage. Most importantly, Bax, cytochrome c, p-p38 MAPK, and p–c-Jun were all upregulated in rats injected with NMDA, but these expression patterns were reversed by continuous Lutein uptake. Bcl-2, p-GSK-3b, and p-Akt in the Lutein-treated eyes were increased compared with the NMDA group. Lutein has neuroprotective effects against retinal damage, its protective effects may be partly mediated by its anti-excitability neurotoxicity, through MAPKs and PI3K/Akt signaling, suggesting a potential approach for suppressing retinal neural damage.
& Daxiang Lu [email protected] 1
Key Laboratory of State Administration of Traditional Chinese Medicine of China, Department of Pathophysiology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong Province, China
Keywords Lutein Apoptosis MAPKs signaling PI3K/ Akt signaling Retinal ganglion cell Abbreviations RGCs Retinal ganglion cells ERG Electroretinograms phNR Photic negative response RIPA Radio immunoprecipitation assay SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis PVDF Polyvinylidene fluoride ECL Electrochemiluminescence AkT Protein kinase B/PKB ERK Extracellular signal-regulated kinase GSK-3b Glycogen synthase kinase-3b MAPK Mitogen-activated protein kinases
Introduction Lutein is a xanthophyll dietary carotenoid that is structurally similar to zeaxanthin (Sies et al. 1992). Lutein has a chemical formula of C40H56O2 with a long carbon chain with alternating single and double carbon–carbon bonds with attached methyl side groups. At both ends of the carbon backbone the molecule contains a cyclohexene structure with an attached hydroxyl group (Granado et al. 2003). The presence of a hydroxyl group at both ends of the molecule distinguishes Lutein from other carotenoids (Kijlstra et al. 2012). The characteristic structure enables Lutein to react more strongly with singlet oxygen than other carotenoids (Tian et al. 2007). Lutein is predominately present in the macular region and acts as an efficient pigment for absorbing high-energy
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blue light. It is also a direct free radical scavenger to prevent macular damage (Johnson 2014). It is anti-apoptotic and plays a neuroprotective role by decreasing oxidative stress (Wang et al. 2013). However, the effect of Lutein on specific cell populations, such as retinal ganglion cells (RGCs), is unknown. Lutein possesses biological antioxidant activity, and it is a major component of carotenoids with non-provitamin A activity (Wang and Lin 2008). We sought to investigate whether Lutein could reverse the excitotoxicity in RGCs. It can inhibit the increased PI3K activity and Akt phosphorylation after oxidative stress (Lee et al. 2006), which provides a basis for us to investigate the role of Lutein in RGC protection. RGCs are neurons that transmit visual signals from the retina to the brain and are the sole output neurons of the retina. RGCs are the main cells expressing N-methyl-D-aspartate receptors (NMDARs) in retinal neurons, which render these cells susceptible to glutamate excitotoxicity (Huang et al. 2014). RGC dysfunction or loss is associated with various optic neuropathies, including glaucoma (Zhang et al. 2014), diabetic retinopathy (Yuan et al. 2014), optic neuritis (Suhs et al. 2014), or Leber’s hereditary optic neuropathy (Gallenmuller and Klopstock 2014). RGC death may occur via a variety of mechanisms involving, for example, reactive oxygen species (Li et al. 2009), excitatory amino acids (Dun et al. 2007), nitric oxide (Rathnasamy et al. 2014), or apoptosis (Dvoriantchikova et al. 2014). Because of RGC susceptibility to different types of damage and because of their importance, it is extremely urgent to find strategies to protect these cells. Neuroprotective therapies that focus on factors leading to RGC degeneration have attracted increasing attention. Studies on the protective effects of carotenoids (including Lutein) against retinal damage have primarily focused on age-related maculopathy (Parisi et al. 2008), but it is necessary to investigate methods to protect RGCs from all mechanisms of damage. According to its properties and function, we believe that Lutein may be the basis for such a therapeutic strategy. The purpose of this study was to examine the effects and possible mechanisms of Lutein on retinal damage in vivo. We studied the effects of Lutein against NMDA-induced retinal damage in rats.
Materials and Methods Animals Female Sprague–Dawley (SD) rats (body weight 200–250 g) were kept under 12-h light/12-h dark conditions. All animal procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National
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Institutes of Health. And we make all efforts to minimize the suffering and number of animals used. Rats were randomly divided into six experimental groups and each group containing 15 rats: a control group, a sham group, NMDA group, NMDA and Lutein (low concentration, middle concentration, and high concentration) group. NMDA-Induced Retinal Damage Model and Treatment with Lutein Retinal damage was induced by N-methyl-D-aspartic acid (NMDA, Sigma, St. Louis, MO, USA) as described by Siliprandi et al. (1992). Briefly, the animals were anesthetized with 10 % chloral hydrate (0.38 ml/100 g; 302170, Kermel, Tian Jing, China) by intraperitoneal injection. Body temperature was maintained at between 37.0 and 37.5 °C with the aid of a heating pad and heating lamp. Retinal damage was induced by the injection (1 ll/ eye) of NMDA dissolved at 40 mM in 0.9 % normal saline (NS), which was injected into the vitreous body of the eyes under anesthesia. One drop of 0.01 % chloramphenicol ophthalmic solution (Yuan Da Pharmaceuticals, Wu Han, China) was applied topically to the treated eye immediately after the intravitreal injection. On the fourth day after the NMDA injection, the rats were employed for visual behavior detection and the next day they were allocated for ERG recording, then the eyeballs were enucleated for immunofluorescence, histological, and Western blot analyses. Lutein (0.25, 0.5, or 1.0 mg/kg,) was dissolved in corn oil immediately before use and intraperitoneally injected five times (0, 1, 2, 3, and 4 days after the NMDA injection). Visual Behavior Detection The visual function of the retina could be evaluated by a black-and-white box, which is formed by two chambers (a white and a black chamber) with a door opening between them (Fig. 1). This system was employed to test the white– black visual discrimination of the rats (Lin et al. 2008). An aperture (10 9 12 cm) between the black-and-white chambers allowed the rats to travel freely from one to the other. The black chamber was illuminated with an infrared light, while the white chamber was illuminated by a bright white light. Two cameras, installed in the two chambers separately, captured the activities of the rats and were connected to a Noldus EthoVision XT 8.0 recorder and monitor. The rats were placed into the middle of the white chamber at the start of the trial and left in the box for 5 min. The number of intercompartmental crosses, the distance moved, the mean velocity, and the time spent in each chamber were recorded by the Noldus EthoVision XT 8.0 software.
Cell Mol Neurobiol Fig. 1 Behavioral assay of rats in the black-and-white box. The red lines represent the moving trajectory of a single rat in the two experimental groups. The representative rats in the control group spent more time traveling in the black chamber than in the white chamber (a), as did the Sham group (b). In the NMDA group, the rats spent almost equal time in the black chamber and in the white chamber (c). Lutein increased the time the animals spent in the black chamber (d, e, f). 0.25L ? N = 0.25 mg/kg Lutein ? NMDA group; 0.5L ? N = 0.5 mg/kg Lutein ? NMDA group; 1.0L ? N = 1.0 mg/kg Lutein ? NMDA group. g, h The statistical graphs of the rats’ time in the black chamber and white chamber. The results are reported as the mean ± SEM. *P \ 0.05 versus the control group, # P \ 0.01 versus the NMDA group, n = 15 (Color figure online)
Electroretinography (ERG) Functional signals and activities of each cell type could be recorded by electroretinography (ERG), and the photopic negative response (PhNR, induced by RGCs) (Chen et al. 2008) was analyzed to determine the change in RGCs after intravitreal injection of NMDA. Rats should be adapted to the dark before ERG recordings to reach a stable status. The time for dark adaptation in the present experiment was at least 70 min (Wang et al. 2011). After stabilization, the ERGs were recorded. In brief, the control rats were anesthetized with 10 % chloral hydrate (0.38 ml/100 g), and the pupils were dilated with tropicamide. Recording electrodes were placed on the corneas. Two reference electrodes were inserted into subcutaneous tissue before the ears, and one ground electrode was inserted into the subcutaneous tissue of the tail, as in a previous study (Zhao et al. 2013). The a-wave, b-wave, and PhNR were recorded using a Roland Consult electrophysiological diagnostic system (Brandenburg, Germany). Scotopic ERGs (a-wave, b-wave; 3.0 and 10.0 cds/m2, white flash) were first
recorded after an 8-h dark adaptation. After light adaption under a continuous blue background (25 cds/m2) for 5 min to suppress rod cell electrical activity, PhNR was recorded with red flashes (3.0 and 10.0 cds/m2). The amplitude of the a-wave was measured from its baseline to its peak, and the amplitude of the b-wave was measured from the trough of the a-wave to the peak of the b-wave. The amplitude of PhNR was defined as the first negative peak following the b-wave that was measured relative to the baseline. PhNR wave was measured using pCLAMP_10.2 software after applying 50 Hz low-pass filtering. Retina Immunofluorescence and the Retinal Whole Mounts The rats were anesthetized by an intraperitoneal injection of 10 % chloral hydrate on the fifth day after intravitreal injection of NMDA (40 mM) at 1 ll/eye. Each eye was enucleated; then, the retina was moved off the eye cup and immersed for 30 min at room temperature in a fixative solution containing 4 % paraformaldehyde dissolved by
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PBS (pH 7.4). After washing six times with PBS, the retina was blocked in 5 % donkey serum in Tris–phosphate buffer with 3 % Triton 100 (PBST) for 1 h at room temperature. Then, the retina was incubated with a primary antibody goat polyclonal antibody against Brn-3 (1:100, Santa Cruz Biotechnology) for 48 h at 4 °C after that the primary antibody was removed and the retina was rinsed six times with PBST (containing 0.3 % Triton 100), then it was incubated with appropriate fluorescent secondary antibody dylight 488-donkey anti-goat IgG (1:1000, EarthOx) for 3 h at room temperature and then the secondary antibody was removed and the retina was rinsed six times with PBST (containing 0.3 % Triton 100) again, at last the antiquenching solution was added to the retina for capturing images using a Leica epifluorescence microscope to count the whole-mounted RGCs with the 1.5 mm interval lattice method, then every lattice field is photographed, after that the RGCs are numbered, then the average number in one lattice field is calculated, at last the density of the RGCs in the whole retina is calculated to make the statistical graph. Western Blot Analysis The rats were anesthetized by an intraperitoneal injection of 10 % chloral hydrate on the fifth day after intravitreal injection of NMDA (40 mM) at 1 ll/eye. The eye was enucleated and the retina was detached, then lysed in icecold RIPA lysis buffer (Beyotime, Nantong, Jiangsu, China) and for full cleavage the retina lysate was broken using the ultrasonic instrument. The lysate was centrifuged for 15 min at 12,000 rpm, 4 °C, and the supernatants were collected for protein analysis. The total protein concentration was determined using an Enhanced BCA Protein assay kit (Beyotime, Nantong, Jiangsu, China). Equal amounts of proteins (40 lg) were then separated by 12 % SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking in Tris–phosphate buffer with 0.1 % Tween 20 (TBST) containing 5 % non-fat milk and washing with TBST, the membranes were incubated with primary antibodies obtained from Cell Signaling Technology (rabbit, 1:1000) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated anti-rabbit antibody (1:5000, Cell Signaling Technology, Inc. Danvers, MA, USA) at room temperature for 2 h. Signals were detected on a gel imaging system using the ECL Western blotting substrate (Millipore, Billerica, MA, USA). Statistical Analysis Statistical analysis was performed with SPSS software, version 19.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) followed by the least
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significant difference multiple comparison test was used. P \ 0.05 was considered significant.
Results Alteration of Visual Behavioral After Intravitreal Injection NMDA in SD Rats Rats prefer dark and narrow places. Therefore, in the blackand-white box test, when a rat is placed in the center of a white chamber, it will enter the black chamber through an aperture between the white and black chamber. Rats stayed in the dark chamber for a longer time than in the white chamber in both the control groups (black duration 229.7 ± 6.7 s; white duration 70.2 ± 6.7 s, Fig. 1a) and the sham group (black duration 220.5 ± 5.8 s; white duration 79.5 ± 6.0 s, Fig. 1b), while the rats spent similar times in the dark and white chambers in the NMDA group (black duration 162.7 ± 6.8 s; white duration 137.3 ± 6.8 s, Fig. 1c). However, the time rats stayed in the dark was extended by Lutein treatment (Fig. 1d–f). There was a significant difference in the duration in the dark chamber between the control group (229.7 ± 6.7 s) and NMDA group (162.7 ± 6.8 s, P \ 0.01, Fig. 1a, c). Compared with the NMDA group, the duration in the dark chamber was extended in the 0.25-mg/kg Lutein group (black duration 187.9 ± 10.0 s; white duration 112.1 ± 10.0 s), the 0.5-mg/ kg Lutein group (black duration 216.3 ± 7.3 s; white duration 83.7 ± 7.3 s), and the 1.0-mg/kg Lutein group (black duration 217 ± 11.3 s; white duration 82.9 ± 11.3 s). Significant differences were observed among the NMDA group, the 0.5-mg/kg Lutein group (216.3 ± 7.3 s), and the 1.0-mg/kg Lutein group (217 ± 11.3 s, P \ 0.01). Different PhNR Amplitude Changes in ERG Recordings After Intravitreal Injection of NMDA PhNR is a negative-going wave that occurs following the b-wave. Because its origin remains undetermined, PhNR is significantly reduced in human patients with primary openangle glaucoma (Huang et al. 2012), anterior ischemic optic neuropathy (Rangaswamy et al. 2004), and other optic nerve neuropathies (Machida et al. 2004), consistent with an origin in ganglion cells or their axons. There was no significant difference in PhNR amplitude between the control group (14.3 ± 1.6 lV) and the sham group (13.3 ± 0.9 lV), but there was a significant difference between the control group (14.3 ± 1.6 lV) and the NMDA group (4.8 ± 0.7 lV; P \ 0.01). Compared with the NMDA group (4.8 ± 0.7 lV), the PhNR amplitude increased in the 0.25-mg/kg Lutein group, in the 0.5-mg/kg
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Lutein group, and in the 1.0-mg/kg Lutein group. A significant difference was observed between the NMDA group (4.8 ± 0.7 lV) and the 0.5-mg/kg Lutein group (12.4 ± 1.8 lV; P \ 0.01), but there were no significant differences among the NMDA group, the 0.25-mg/kg Lutein group (8.0 ± 0.8 lV), and the 1.0-mg/kg Lutein group (7.5 ± 1.3 lV; Fig. 2b). The Change in RGC Number After Intravitreal Injection NMDA by Retinal Whole Mounts On the fifth day, rat eyes were enucleated after sacrifice; then, the retinas were separated from the cup of the eyes and exposed to a primary antibody against Brn-3 to count the RGCs. There was no significant difference in RGC number between the control group (884 ± 30 cells/mm2; Fig. 3a) and the sham group (788 ± 23 cells/mm2; Fig. 3b), but there was a significant difference between the control group (884 ± 30 cells/mm2) and the NMDA group (101 ± 7 cells/mm2; Fig. 3c). Compared with the NMDA group, the number of RGCs was increased in the 0.25-mg/ kg Lutein group (177 ± 13 cells/mm2; Fig. 3d), in the 0.5mg/kg Lutein group (256 ± 16 cells/mm2; Fig. 3e), and in the 1.0-mg/kg Lutein group (162 ± 17 cells/mm2; Fig. 3f). And significant differences among the 0.5-mg/kg Lutein group (256 ± 16 cells/mm2) and the NMDA group (101 ± 7 cells/mm2). Effects of Lutein on Expression of Bax, Bcl-2, Caspase-3, and Cytochrome c in NMDA-Induced Retinas To study the role of Lutein in NMDA-induced retina injury, we analyzed the levels of apoptosis markers in the retinas using Western blotting. Cleaved caspase-3, the
activated form of caspase-3, was clearly upregulated in the NMDA group compared with the control group. The same was true of the pro-apoptotic proteins Bax and cytochrome c (P \ 0.05; Fig. 4). In contrast, the anti-apoptotic protein Bcl-2 was clearly increased in the 0.5-mg/kg Lutein group, and the ratio of Bax to Bcl-2 significantly declined in the 0.5-mg/kg Lutein group compared with the NMDA group (P \ 0.05; Fig. 4). Effect of Lutein on Expression of ERK, p38 MAPK, and c-Jun in NMDA-Induced Retina Damage ERK, p38 MAPK, and c-Jun were also analyzed by Western blot. Phosphorylated p38 MAPK and c-Jun were significantly elevated in the NMDA group compared with the control group (P \ 0.05). These increases were inhibited by constant Lutein intake, especially 0.5 mg/kg Lutein, compared with the NMDA group (P \ 0.05; Fig. 5). Phosphorylated ERK had almost no change in all the groups. Effect of Lutein on Expression of Akt and GSK-3b in NMDA-Induced Retinas To clarify the effect of Lutein on Akt activation in NMDAinduced retina damage, we performed immunoblot analyses of phosphorylated (activated) Akt. The activation of Akt in the retina was significantly increased by Lutein intake, particularly the 0.5-mg/kg dose (Fig. 6), compared with the NMDA group (P \ 0.05). GSK-3b, a downstream protein of Akt, was significantly increased in the 0.5-mg/kg Lutein group (Fig. 6) compared with the NMDA group (P \ 0.05). There were no significant difference in total Akt or total GSK-3b between groups (Fig. 6; n = 5).
Fig. 2 The effects of Lutein on NMDA-induced changes in ERG in SD rats. a The normal ERG. There was an obvious PhNR wave. b Statistical analysis of the mean amplitudes of the PhNR waves of photopia 3.0. The PhNR wave amplitude of ERG was significantly lower in the NMDA group, but the effects induced by NMDA were largely reversed by Lutein (0.5 mg/kg). The data in all bar graphs are shown relative to controls. The data represent the mean ± SEM. *P \ 0.05 versus control; #P \ 0.01 versus the NMDA group, n = 13
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Cell Mol Neurobiol Fig. 3 The effects of Lutein on NMDA-induced change in the number of RGCs in SD rats. The number of RGCs in the NMDA group (c) was significantly reduced compared with control (a) using Brn-3-marked cells. Lutein (d, e, f) increased the number of Brn-3-positive cells in retina, especially 0.5 mg/kg Lutein (e). 0.25L ? N = 0.25 mg/kg Lutein ? NMDA group; 0.5L ? N = 0.5 mg/kg Lutein ? NMDA group; 1.0L ? N = 1.0 mg/kg Lutein ? NMDA group. (g) Statistical analysis of the mean number of RGCs. The number of RGCs was significantly reduced in the NMDA group, but the effects induced by NMDA were largely reversed by Lutein (0.5 mg/kg). The data in all bar graphs are shown relative to controls. The data represent the mean ± SEM. *P \ 0.05 versus control; #P \ 0.01 versus NMDA group, n = 9
Discussion Excitability neurotoxicity was common cause of many ocular diseases, which lead to irreversible RGC damage and RGCs are sensitive to excitability neurotoxicity in pathological situations in vivo and in vitro (Yamasaki et al. 2005). In pathologies such as ischemia/reperfusion, glaucoma, and DR, the overproduction of ROS, free radicals, and excitatory amino acids overwhelm the intrinsic eliminating mechanism (Osborne et al. 2004; Tezel 2006), and these events exceed the ability of the normal retina to protect against excitability neurotoxicity which leads to RGC damage. NMDA-induced apoptosis in RGCs is caspase independent (Kim et al. 2013) and yet includes the Akt/PI3K, the p38 MAPK, and the ERK signaling
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pathways in previous study (Nakazawa et al. 2005; Manabe and Lipton 2003; Lin et al. 2007). In our experiment we investigated the influence of Lutein on NMDA-induced retina excitability neurotoxicity using a combination of the retina whole-cell mount, immunohistochemistry, ERG, visual behavior detection, and Western blotting. We found significant improvement in the RGCs treated with Lutein compared with the injury group, indicating that Lutein improved the ability of RGCs to avoid excitability neurotoxicity. Lutein blocks excitability neurotoxicity, oxidative stress, inflammation, and apoptosis, and the agent is neuroprotective against the NMDA-induced injury (Miyake et al. 2014; Sun et al. 2014; Woo et al. 2013; Li et al. 2009, 2012a, b). The mechanism of Lutein’s regulation of cell damage and
Cell Mol Neurobiol Fig. 4 The expression of apoptotic proteins Bax, Bcl-2, caspase-3, and cytochrome c in NMDA-induced retina injury by Western blot. a The Bax and Bcl-2 protein expression in NMDA-induced retina. The ratio of Bax/Bcl-2 was significantly increased in the NMDA group compared with the control group, but this was reversed by Lutein uptake. b The caspase-3 protein expression in NMDA-induced retina. The ratio of cleaved caspase-3/caspase-3 was significantly elevated in the NMDA group compared with the control group, but this was also reversed by Lutein uptake. c The cytochrome c protein expression in NMDA-induced retina. The expression of cytochrome c was significantly increased in the NMDA group compared with the control group, but this was again reversed by Lutein uptake. The results are reported as the mean ± SEM. #P \ 0.01 versus the control group, *P \ 0.01 versus the NMDA group (n = 6)
apoptosis is through the members of the Bcl-2 family, the MAPK pathway (Lo et al. 2012, 2013), and the PI3K/Akt pathway. The Bcl-2 family consists of both apoptosis promoters, including Bax and Bad, and apoptosis inhibitors, which include Bcl-2, Bcl-X, and Mcl-1. A family of aspartate-specific proteases termed caspases is activated in the execution phase of apoptosis. The cleavage of caspase-3 is often viewed as the final step of the process that promotes the end of the apoptotic signaling pathway. In addition, the ratio of Bax/Bcl-2 might determine survival or death (Verma et al. 2013). In vivo, we found that Lutein improved the response to light of NMDA-damaged rats in the black-andwhite box assay (Fig. 1), increased the PhNR wave amplitude of NMDA-damaged rats according to ERG recordings (Fig. 2), and rescued the RGC decrease induced by NMDA as shown by whole-cell mount technology (Fig. 3). Then, we found that the expression of Bax, cleaved caspase-3, and cytochrome c was inhibited by Lutein, while the expression of Bcl-2 was promoted by Lutein. The middle dose (0.5 m/ kg) of Lutein had the optimal effect (Fig. 4), which was in
agreement with a previous finding (Han et al. 2012) and (Chen et al. 2012). The MAPKs is a kind of serine or threonine protein kinase, a major signaling pathway in cellular stress and damage response, and the MAPK signaling pathway is believed to regulate the apoptosis of retina cells after ischemia/reperfusion damage (Ishizuka et al. 2013). The kinase family has three members in the classical pathway, including extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and the protein kinase p38 (Xu et al. 2014). ROS-activated ERK contribute to synaptophysin degradation and neuronal dysfunction in the diabetic retina (Sasaki et al. 2010), while JNK activation has the opposite effect. The effect of p38 on apoptosis remains controversial (Ishizuka et al. 2013; Li et al. 2012). Our data agree with these. In the present investigation, we found that the activation of p38 MAPK and c-Jun were suppressed by Lutein, indicating that Lutein protects against NMDA-induced RGC damage through the MAPK pathway (Fig. 5).
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Fig. 5 Western blots of phosphorylated ERK, p38 MAPK, and c-Jun in NMDA-induced retina injury. a Phosphorylated ERK in NMDAinduced retina. b Phosphorylated p38 MAPK was higher in the NMDA group compared with the control group, but Lutein reversed
this. c Phosphorylated c-Jun was higher in the NMDA group compared with the control group, but Lutein reversed this. The results are reported as mean ± SEM. #P \ 0.01 versus the control group, *P \ 0.01 versus the NMDA group (n = 5)
The serine/threonine kinase Akt, also named protein kinase B (PKB), plays a critical role in many cellular activities, including cell survival, growth, proliferation, angiogenesis, metabolism, and migration (Manning and Cantley 2007). Phosphorylated Akt has a neuroprotective effect in a diabetic rat model (Park et al. 2014). We also found that Lutein increased Akt activation in NMDA-induced retina damage, and p-Akt activated its downstream protein GSK-3b to carry out its neuroprotection function (Fig. 6). Taken as a whole, our data demonstrate that Lutein exerts anti-apoptotic effects during NMDA-induced RGC injury by activating Akt and inhibiting the phosphorylation of p38 MAPK and c-Jun, which result in increased Bcl-2 levels and reduced levels of Bax, caspase-3, and
cytochrome c. Thus, our results provide important insights into the potential mechanisms involved in the neuroprotective effects of Lutein, which may make it a candidate for a neuroprotective agent against ocular damage. However, we found that the neuroprotective effects of Lutein were not completely in a dose-dependent manner during NMDA-induced RGC injury, and the middle concentration of Lutein (0.5 mg/kg) played a better effect, which may be due to the high concentration of lutein in vivo conversion of other carotenoids, then competed with Lutein to bind to lutein-binding protein, resulting in the decreased retinal Lutein content. Research shows that the increasing of Lutein in retina could reduce light-induced photoreceptor apoptosis while the increasing of Lutein in the serum does not have the effect (Thomson et al. 2002). Further studies
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Cell Mol Neurobiol Fig. 6 Western blots of phosphorylated Akt and GSK3b in NMDA-induced retina damage. a The p-Akt/Akt ratio in NMDA-induced retina was increased, but this was reversed in the Lutein group. b The p-GSK-3b/GSK-3b ratio was significantly increased in the NMDA group, but this was also reversed in the Lutein group. The results are reported as the mean ± SEM. #P \ 0.01 versus the control group, *P \ 0.01 versus NMDA group (n = 5)
are needed to elucidate the exact mechanism by which Lutein protects RGCs. Acknowledgments This work was supported by grants from the National Program on Key Basic Research Project (973 Program No. 2011CB707501), and the National Natural Science Foundation of China (Nos. 81371442 and 81471236). Compliance with Ethical Standards Conflict of interest No conflicting relationship exists for any author.
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