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reperfusion injury by inhibiting neuronal apoptosis in mice
Neurochemistry International 61 (2012) 649–658
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Glycine attenuates cerebral ischemia/reperfusion injury by inhibiting neuronal apoptosis in mice Yan Lu a,b, Jing Zhang a,b, Bingqing Ma a, Kexue Li a,b, Xiaoyu Li a, Hui Bai b, Qing Yang b, Xudong Zhu a,b, Jingjing Ben a,b, Qi Chen a,b,⇑ a b
Atherosclerosis Research Centre, Laboratory of Molecular Intervention with Cardiovascular Diseases, Nanjing Medical University, Nanjing 210029, PR China State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, PR China
a r t i c l e
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Article history: Received 5 March 2012 Received in revised form 18 June 2012 Accepted 3 July 2012 Available online 10 July 2012 Keywords: Glycine Cerebral ischemia/reperfusion Apoptosis SH-SY5Y cell siRNA Neurons Glycine receptor
a b s t r a c t Glycine is a cytoprotector to protect cells against ischemic damage by counteracting neuronal depolarization. However, whether it can directly inhibit neuronal apoptosis is unknown. In this study, we demonstrated that glycine could attenuate ischemia/reperfusion (I/R) induced cerebral infarction and improved neurological outcomes in mice. The protective effect of glycine was associated with reduction of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) positive cells, deactivation of phosphor-JNK, inhibition of caspase-3 cleavage, down-regulation of FasL/Fas, and up-regulation of bcl-2 and bcl-2/bax in the mouse I/R penumbra. The beneficial effect of glycine against oxygen and glucose deprivation (OGD) induced injury was also confirmed in SH-SY5Y cells as well as in primary cultured neurons, which was significantly dampened by knockdown of glycine receptor a1 (GlyR a1) with siRNA transfection or by preventing glycine binding with glycine receptor using a specific antibody against glycine receptor. These results suggest that glycine antagonize cerebral I/R induced injury by inhibiting apoptosis in mice. Glycine could block both extrinsic and intrinsic apoptotic pathways for which GlyR may be required. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Stroke is a leading cause of death and adult disability worldwide. Of all strokes, ischemic stroke accounts for approximately 80% (Feigin et al., 2003; Liu et al., 2010). Thrombolysis and revascularization of the obstructed blood vessels are effective and safe therapies to restore the cerebral blood flow. However, the efficacy of these interventions is satisfactory in a selected few types of stroke and is limited by a narrow time window (usually in the very early period) (Hankey and Warlow, 1999; Kwiatkowski et al., 1999). Thus, finding more effective therapeutic neuroprotective agents has become a priority in the field. Abbreviations: CNS, central nervous system; ERK1/2, extracellular signal-regulated kinase 1 and 2; GlyRs, glycine receptors; GlyR a1, glycine receptor a1; IACUC, institutional animal care and use committee; I/R, ischemia/reperfusion; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MCAO, middle cerebral artery occlusion; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; NMDA, N-methyl-D-aspartate; OGD, oxygen and glucose deprivation; TTC, 2,3,5triphenyl tetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. ⇑ Corresponding author. Address: Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, PR China. Tel.: +86 25 86862610; fax: +86 25 86508960. E-mail address: [email protected] (Q. Chen). 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.07.005
Inhibitory amino acid taurine and L-serine can protect the brain from ischemic injury. It is supposed to be mediated by activating glycine receptors (GlyRs) (Wang et al., 2007, 2010). GlyRs, as well as glycine-containing fibers and cell bodies, are widely distributed in brain rather than restricted to the spinal cord and brain stem (Legendre, 2001). As a major inhibitory neurotransmitter, glycine can activate GlyR to reach chloride influx induced postsynaptic hyperpolarization and reduces neuronal excitability. This antiexcitotoxic property is by counteracting neuronal depolarization and subsequent cascade of biochemical events that would result in cell death (Wang et al., 2010). Glycine also exhibits a directive protection against ischemia induced injury to organs and tissues (Yin et al., 2002; Zhang et al., 2003; Omasa et al., 2003; Tang et al., 2006). We showed that cytoprotection against ATP depletion by glycine is mediated by GlyR (Pan et al., 2005). The extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase and AKT pathways constitute GlyR-coupled signaling pathways (Jiang et al., 2010), suggesting a potential anti-apoptotic property should be involved in cytoprotection of glycine. Apoptosis occurs in the delayed neuronal death in the ischemic lesions (Kametsu et al., 2003; Waldmeier, 2003). In order to clarify if glycine antagonizes ischemic injury by an anti-apoptotic mechanism in the brain, we used an experimental transient middle cerebral artery occlusion
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(MCAO) model in mice. Our results demonstrated that glycine may attenuate mouse cerebral ischemia/reperfusion (I/R) injury by inhibiting cell apoptosis. 2. Materials and methods 2.1. Animals Male ICR mice were supplied by the Experimental Animal Center of Nanjing Medical University, Nanjing, China. The experimental protocol was approved by Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University. All experiments were performed in accordance with international standards on the ethical treatment of animals. Mice were housed under climatecontrolled conditions with a 12-h light/dark cycle and provided standard food and water ad libitum. Glycine (Sigma, St. Louis, MO, USA) was dissolved in physiological saline and was intraperitoneally injected into mice 30 min before MCAO for the pretreatment study. All MCAO mice were divided into receiving vehicle, low-dose glycine (250 mg/kg), and high-dose glycine (1000 mg/ kg) groups. For the posttreatment experiments, drug treatments were given via intraperitoneal injection 10 min after reperfusion.
age analyzer. Infarct volume of each slice was obtained by multiplying the infarct area by 2-mm thickness. Total infarct volume was determined by summing up infarct volume of five consecutive slices. All brain slices were analyzed for their infarct volume using the Image-J analysis software. Percentage infarct volume was calculated as follows: [(VC VL)/VC] 100, where VC is the volume of control hemisphere (Right side), VL is the volume of non-infarcted tissue in the lesioned hemisphere (Right side). 2.4. TUNEL staining Samples from sham-operated, vehicle, and glycine-high groups were used for experiments. Mice were deeply anesthetized with chloral hydrate 72 h after reperfusion. After transcardiac perfusion with 50 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4), brains were removed and stored in the same paraformaldehyde solution overnight. Multiple, paraffin-embedded, coronal sections (4 lm thick) were taken from brain and stained by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. The apoptotic neurons were counted using an inverted microscope (Olympus BX51; Japan) in the peri-infarct cortex (three fields were counted in each case at 400 magnification) in a blinded manner.
2.2. MCAO model
2.5. Western blot analysis
Transient focal cerebral ischemia was induced by right MCAO with a modified intralumenal filament technique (Hata et al., 1998). Briefly, mice were anesthetized by 3.5% chloral hydrate (1 mg/kg). A 5-0 monofilament surgical nylon suture with a heatblunted tip was introduced into the left internal carotid artery through the stump of the external carotid to the base of the right to stop blood flow. After 90 min, the filament was withdrawn to allow blood reperfusion. Meanwhile the ipsilateral common carotid artery remained ligated, the neck skin was closed, and the skin was sutured. Rectal temperature, monitored with a digital thermometer inserted 2 cm into the anus, was maintained at 37– 38 °C throughout the operation using a feed back-regulated heating blanket. All animals recovered from anesthesia within 30 min of wound closure. Sham-operated animals underwent the same procedure except for MCAO.
The right ischemic cortex in the middle cerebral artery region (middle cerebral artery cortex) was obtained from preoperation (healthy control) and 24 h after MCAO. Protein concentration was determined using BCA protein assay kit (PIERCE, Rockford, IL, USA). Individual samples (60 lg each) were separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, USA). Immunoblotting was performed by incubation of the membrane in 5% dry milk for 1 h and in the specific antibody overnight at 4 °C. Rabbit antibodies against P-JNK, caspase-3, bcl-2 and bax were obtained from Cell Signaling Techonology (Beverly, MA, USA). Rabbit anti-FasL antibody was obtained from Abcam (Cambridge, UK). Rabbit anti-GlyRa1/2 antibody was obtained from Abd serotec (Oxford, UK). After washing twice with Tris-buffered saline-Tween 20, each membrane was incubated with a secondary antibody (1:5000) for 2 h. Cellular b-actin or GAPDH was used as a loading control to prove that all of the lanes were loaded with the same amount of protein. The membranes were then developed using an enhanced chemiluminescence system. The figures were analyzed with Image J.
2.3. Measurement of the area of early ischemic brain injury Mice were examined for neurological deficit using a four-tiered grading system before giving anesthesia, 24 h, and 72 h after MCAO and reperfusion (Connolly et al., 1996a,b). A score of 1 was given if the animal was demonstrated normal spontaneous movements; 2 was given if the animal was noted to be turning to the right (i.e., clockwise circles) when viewed from above (i.e., toward the contralateral side); 3 was given if the animal was observed to spin longitudinally (clockwise when viewed from the tail); and 4 was given if the animal was crouched on all fours, unresponsive to noxious stimuli. This scoring system is based upon similar scoring systems used in rats (Bederson et al., 1986; Menzies et al., 1992) which are based upon the contralateral movement of animals with stroke. After cerebral infarction, the contralateral side is ‘‘weak’’ and so the animal tends to turn toward the weakened side. Mice were killed by decapitation 24 h or 72 h after MCAO. Brains were removed and chilled in ice-cold saline for 5 min. Four 2-mm consecutive coronal slices were made by using a brain slicer, beginning from the anterior pole. Slices were incubated in saline solution containing 2% 2,3,5-triphenyl tetrazolium chloride (TTC) (Sigma, St. Louis, MO, USA) at 37 °C for 30 min. They were then fixed by 10% formalin neutral buffer solution (pH 7.4) for 1 h. Infarct area in each slice was evaluated by scanned digital images with an im-
2.6. RNA extraction and real-time PCR Total RNA was isolated using Trizol reagent (Invitrogen, San Diego, CA, USA) in accordance with the manufacturer’s protocol. After extraction, 0.5 lg of total RNA was used as template to synthesize cDNA using a first strand synthesis kit (Invitrogen, San Diego, CA, USA). The cDNA from this synthesis was then used in quantitative real-time PCR analysis (ABI-Prism 7700 sequence detection system; Applied Biosystems, Foster City, CA, USA) using SYBR Green dye. The following primer pairs for murine were used: Fasl, 50 TGAATTACCCATGTCCCCAG-30 (forward) and 50 -AAACTGACCCTGGAGGAGCC-30 (reverse); Fas, 50 -TGGCAGAGGAGCCTAGTTGT-30 (forward) and 50 -CACACCCAGGAACAGTCCTT-30 (reverse); bcl-2, 50 -ATGATAACCGGGAGATCGTG-30 (forward) and 50 -GTTCAGGTACTCAGTCACC-30 (reverse); bax, 50 -ACCAGCTCTGAACAGATCATG-30 (forward) and 50 -TGGTCTGGATCCAGACAAG-30 (reverse); actin, 50 TTCGTTGCCGGTCCACA-30 (forward) and 50 -ACCAGCGCAGCGATATCG-30 (reverse) (Pinkoski et al., 2002; Guo et al., 2003; Symonds et al., 2005; Yoshimatsu et al., 2009).
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2.7. Oxygen-glucose deprivation We used oxygen and glucose deprivation (OGD) reoxygenation, an in vitro model that best mimics in vivo cerebral ischemia reperfusion, in the study. Cells were washed once. During OGD, the medium was changed in a HEPES-buffered glucose-free medium (pH 7.4) containing 154 mmol/L NaCl, 5.6 mmol/L KCl, 2.3 mmol/L CaCl2, 1.0 mmol/L MgCl2, 3.6 mmol/L NaHCO3, and 5 mmol/L HEPES. OGD was induced by incubating cells in this medium and placing cells in an anaerobic chamber with an atmosphere of 95% N2 and 5% CO2 at 37 °C (Liu et al., 2010). OGD was terminated after 12 h by replacing the glucose-free medium back to DMEM under normal conditions for another 24 h as an injury reperfusion model in the following experiments. Control cells were maintained normally. Glycine was added to the culture medium before OGD treatment and throughout the OGD reperfusion.
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2 mM glutamine. The medium were replaced with fresh medium every 3 days. Experiments were performed on days 7–10. Neurons were pre-treated for 1 h with 10 lg/ml, 2 lg/ml antibody against GlyR, 10 lg/ml normal rabbit IgG, or pre-treated for 30 min with 10 lM MK-801 before OGD damage. Neurons were subjected to OGD for 3 h followed by 24 h of reoxygenation. Glycine was added to the culture medium before OGD treatment and throughout the OGD reperfusion.
2.12. Statistical analysis All values are presented as mean ± standard error. Data of two groups were analyzed for statistical significance with Student’s ttest. Multiple comparisons were made by using one-way ANOVA. Differences between means were considered significant if p < 0.05.
2.8. siRNA transfection 3. Results Transfections were performed by using Lipofectamine 2000 (Invitrogen, San Diego, CA, USA). The target sequence in human GlyR a1 gene was as follows: GGCCUAUAAUGAAUACCCUtt and AGGGUAUUCAUUAUAGGCCag. A scrambled siRNA was used as a negative control. All oligonucleotides were obtained from Ambion RNA. SH-SY5Y cells were seeded 1 day prior to transfection. They were 30% confluent when they were transfected with 25 nM positive or scrambled oligonucleotides in Lipofectamine 2000 and Opti-MEM (Invitrogen, San Diego, CA, USA) without serum for 72 h (Zhu et al., 2011). Expression of glycine receptor a1 was estimated by western blot using an antibody against GlyR a1. 2.9. Measurements of cell damage Cellular 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed by addition of 25 ll MTT stock solution (5 mg/ml) to each well. After incubation for 4 h, the MTT solution was carefully decanted off and formazan was dissolved with 200 ll DMSO. The absorbance was measured at 492 nm using a 96-well plate ELISA reader (CliniBio 128, Austria).
3.1. Glycine attenuates cerebral I/R injury in mice To test the effect of glycine on cerebral ischemia in mice, infarct volume and neurological score were recorded both 24 and 72 h after MCAO injury. The neurological score before reperfusion was not significantly different among the groups (data not shown). However, the infarct volume was significantly diminished (Fig. 1A and B) and the neurological score was improved by glycine administration 24 h after MCAO (Fig. 1C). The neuroprotective effects of glycine were also found in mice 72 h post MCAO injury (Fig. 1D–F). Treatment with a high dose of glycine reduced significantly total infarct volume comparing with the vehicle group both 24 h (48.86 ± 3.9 mm3 versus 64.66 ± 2.58 mm3, Fig. 1B) and 72 h (49.02 ± 2.2 mm3 versus 65.42 ± 3.4 mm3, Fig. 1E) after MCAO. Consistently, a significant improvement in neurological score was observed in glycine-high groups comparing with vehicle groups both 24 h (2.59 ± 0.54 versus 1.86 ± 0.23, Fig. 1C) and 72 h (2.88 ± 0.79 versus 2 ± 0.5, Fig. 1F) after MCAO. These results suggest that glycine may attenuate cerebral I/R injury in mice.
2.10. LDH measurement
3.2. Glycine inhibits I/R-induced neuronal apoptosis in mice
Cell injury was determined by measuring the amount of lactate dehydrogenase (LDH) released into the medium (Koh and Choi, 1987). Cultures were exposed to OGD and treated with glycine for 12 h followed by 24 h reoxygenation. The medium was collected before cell was permeabilized with equal volume of 0.4% (v/v) Triton. The activities of extracellular and intracellular LDH were measured separately. The extracellular LDH activity was divided by the total LDH activity (extracellular activity plus intracellular activity) to calculate the percentage of LDH release.
To determine if the neuroprotective effect of glycine is by inhibiting apoptosis, we analyzed penumbra apoptotic status with TUNEL staining. No TUNEL-positive neurons were detected in the sham group (Fig. 2A) but they were evident in vehicle group 72 h after MCAO. Number of TUNEL-positive neurons was significantly decreased by treatment with glycine (Fig. 2B). Furthermore, we checked expression levels of phosphorylated c-Jun N-terminal kinase (JNK) and cleavaged caspase-3 in the middle cerebral artery cortex by western blot. As shown in Fig. 3A and B, MCAO activated JNK phosphorylation 24 h post-MCAO. Glycine treatment could dampen it significantly. Effect of high dose of glycine was much stronger than that of low dose of glycine. Level of phosphorylated JNK in glycine-high group was even lower than that of sham group but was not statistically significant (sham: 127.79 ± 11.7%; vehicle: 170.01 ± 7.2%; glycine: 135.42 ± 8.9%; glycine-high: 89.26 ± 19.1%, Fig. 3B). The cleaved caspase-3 expression 72 h post-MCAO (Fig. 3C and D) was significantly increased in the vehicle groups. They were lower in glycine and glycine-high groups compared with that of vehicle group. Difference between vehicle and glycine-high group was statistically significant (sham: 38.05 ± 5.4%; vehicle: 77.78 ± 8.5%; glycine: 55.9 ± 3.6%; glycinehigh: 33.07 ± 11.2%, Fig. 3D).
2.11. Neuronal culture and treatment Neurons were isolated as previously described (Ming et al., 2006). Briefly, cerebral cortices were dissected from postnatal day 0 ICR mice and incubated in 0.125% trypsin for 30 min at 37 °C. Tissues were then triturated with fire-polished glass pipettes and plated on poly-L-lysine-coated plates or dishes at a density of 1 105. Neurons were cultured with DMEM/F12 (Invitrogen, San Diego, CA, USA) (1:1) supplemented with 10% fetal bovine serum. Cells were maintained in incubators at 37 °C under 5% CO2 atmosphere. After 24 h, the culture medium was changed to neurobasal medium supplemented with 2% B27 (both by Gibco, NY, USA) and
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Fig. 1. Neuroprotective effect of glycine against cerebral ischemic/reperfusion injury in mice. Mice were treated with glycine (low-dose, 250 mg/kg, n = 10; high-dose, 1000 mg/kg, n = 10) or vehicle (n = 8). Sham-operated animals (sham, n = 5) underwent the same procedure except for MCAO. Mice were examined for neurological deficit or killed to measure the infarct volume after MCAO. (A) Representative photos of the mouse brain 24 h after MCAO. (B) Quantitative analysis of total infarct volume in mice 24 h after MCAO. (C) Neurological score 24 h after MCAO. (D) Representative photos of the mouse brain 72 h after MCAO. (E) Quantitative analysis of total infarct volume in mice 72 h after MCAO. (F) Neurological score 72 h after MCAO. Values are shown as the mean ± SE; ##P < 0.01 versus sham group; ⁄P < 0.05 versus vehicle group, ⁄⁄P < 0.01 versus vehicle group.
3.3. Glycine up-regulates bcl-2 and bcl-2/bax and down-regulates FasL/Fas after transient MCAO in mice
3.4. Knockdown of glycine receptor diminishes glycine protection against OGD injury in SH-SY5Y cells
Having established that glycine can markedly inhibit JNK phosphorylation and caspase-3 cleavage in the I/R mouse brain, we wondered which apoptotic pathways were targeted by glycine in vivo. Bcl-2/bax is a marker of intrinsic apoptotic pathway and FasL/Fas is an extrinsic apoptotic pathway marker. We found that bcl-2/bax mRNA ratio was decreased in the focal brain after I/R injury. Glycine treatment resulted in a significant increase in bcl-2/ bax compared with vehicle group (Fig. 4A). In accordance with it, western blot analysis confirmed that there was no significant difference in levels of bcl-2, bax, and bcl-2/bax between sham and vehicle group 24 h after MCAO. Dramatic increases in bcl-2 (sham: 106.89 ± 18.6%; vehicle: 115.41 ± 7.9%; glycine-high: 174.81 ± 20%, Fig. 5B) and bcl-2/bax proteins (sham: 0.88 ± 0.1; vehicle: 1.03 ± 0.1; glycine-high: 1.5 ± 0.2, Fig. 5E) were found when glycine treatment was performed. But glycine did not influence bax expression significantly (Fig. 5C and D). We also found that both mRNA and protein levels of FasL in the infarct middle cerebral artery region were significantly increased after treatment with glycine but not with saline (Figs. 4B and 5F and G). Coincidentally, Fas mRNA level was also significantly increased after I/R which was dampened by treatment with glycine in mice (Fig. 4C). These data indicated that glycine may target at both bcl-2/bax and FasL/Fas mediated apoptosis to attenuate cerebral I/R injury in mice.
To further investigate mechanism underlying glycine’s neuroprotection, we used an OGD cell model in vitro. MTT activity in SH-SY5Y cells was significantly decreased after 12 h OGD followed by 24 h reperfusion. As shown in Fig. 6, treatment with glycine could antagonize the OGD injury in cells obviously. It is dosedependent from 1 to 20 mM. We used 5 mM as a working dose for glycine treatment in vitro in the following experiments. Glycine is a ligand of GlyR. To clarify if GlyR is requisite for neuroprotection of glycine, GlyR knockdown was performed by RNA interference. When GlyR a1 protein expression was knocked down by 70% in SH-SY5Y cells, 71% of recovering MTT effect by glycine was abolished in OGD cells (Fig. 7A–C). Meanwhile, knockdown of GlyR a1 led to a significant increase in LDH release (by 60%) from cells even in the presence of glycine (Fig. 7D). These data revealed that GlyR a1 may be requisite for cytoprotection of glycine against OGD injury in SH-SY5Y cells. 3.5. Glycine increases cell viability and prevents cell death induced by OGD in primary cultured neurons We also observed cytoprotective effect of glycine in primary cultured neurons. The release of LDH from cells occurs with the loss of plasma membrane integrity, a process most often associated with necrotic cell death. Fig. 8A showed that OGD treatment re-
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Fig. 2. TUNEL-staining cells 72 h after MCAO in mice. (A) Representative photomicrographs of TUNEL-positive cells in ischemic penumbra. (B) Quantitative analysis of TUNEL-positive neurons in ischemic penumbra. Values are the mean ± SE (n = 5); ##P < 0.01 versus sham group; ⁄⁄P < 0.01 versus vehicle group.
Fig. 3. JNK phosphorylation and cleaved caspase-3 in the infarct middle cerebral artery region in mice. The left ischemic middle cerebral artery cortex in mice was obtained 24 h after MCAO for measurement of JNK phosphorylation and 72 h after MCAO for measurement of cleaved caspase-3 separately (n = 4, respectively). (A) Representative western blots of phospho-JNK. (B) Quantitative analysis of JNK phosphorylation. (C) Representative western blots of cleaved caspase-3. (D) Quantitative analysis of cleaved caspase-3. The band density values were calculated as a ratio of b-actin. Values are the mean ± SE; #P < 0.05 versus sham group; ⁄P < 0.05 versus vehicle group.
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Fig. 4. mRNA levels of bcl-2/bax, FasL, and Fas in the infarct middle cerebral artery region in mice 24 h after MCAO. (A) Ratio of bcl-2/bax mRNA levels (n = 5). (B) FasL mRNA levels (n = 5). (C) Fas mRNA levels (n = 5). Values are the mean ± SE; #P < 0.05 versus sham group; ##P < 0.01 versus sham group; ⁄⁄P < 0.01 versus vehicle group.
sulted in a significant increase in LDH level compared with control. Glycine 5 mM blocked OGD-induced LDH release by 50%, which was inhibited by pre-treated with an antibody against GlyR. Furthermore, we checked expression levels of cleaved caspase-3 in OGD-insulted neurons. As shown in Fig. 8B and C, OGD treatment led to an increase in cleaved caspase-3 which was dampened in the presence of glycine. Addition of an antibody against GlyR mostly abolished the neuroprotection of glycine (control: 41.21 ± 11.2%; OGD: 253.02 ± 25.4%; glycine: 112.63 ± 8.9%; antibody: 253.22 ± 45.1%; antibody + glycine: 247.4 ± 30%, Fig. 8C). As a co-agonist of glutamate N-methyl-D-aspartate (NMDA) receptor, the neuroprotection of glycine has been thought to be mediated by NMDA receptor (Liu et al., 2007). We used MK-801, a NMDA receptor antagonist to test the role of NMDA receptor in cytoprotection. Fig. 8D showed that treatment with MK-801 alone provided a significant 60% reduction of LDH release compared with OGD. This effect was increased by simultaneous treatment with glycine but there was not a statistical significance between these two groups. The antibody against glycine receptor did not influence the LDH release in MK-801 and MK-801 plus glycine groups, suggesting that GlyR should not be involved in the MK-801’s effect. 4. Discussion Glycine is one of the main components mediating fast inhibitory neurotransmission in the central nervous system (CNS), in which the strychnine-sensitive GlyR, a ligand-gated anionic channel, is primarily involved in it. Besides this neurotransmitter function, glycine has been shown to antagonize shock and ischemia/reperfusion injury in many other non-neuronal tissues and organs including liver, kidney, heart, intestine, and skeletal muscle
(Gundersen et al., 2005; Qi et al., 2007). The protective mechanism of glycine seems vary in cell types. In renal cells, hepatocytes, and endothelial cells, glycine exerts its cytoprotective effect by stabilizing porous defects in the ischemic plasma membranes (den Eynden et al., 2009). In lung ischemia-reperfusion injury, glycine attenuates I/R injury by reducing oxidative damage and suppressing apoptosis (Omasa et al., 2003). In hepatic ischemia, glycine can also prevent apoptosis-related cell death (Duenschede et al., 2006). We demonstrated that glycine could antagonize cerebral I/R injury by suppressing neuronal apoptosis in mice. As a co-agonist of glutamate N-methyl-D-aspartate (NMDA) receptor, glycine can induce neurotoxicity in organotypic hippocampal slice cultures (Sua´rez et al., 2005). Accordingly, compounds blocking the glycine site on the NMDA receptor have been widely investigated. Gavestinel is a one of the selective antagonists for the glycine site on the NMDA receptor. However, no effect of gavestinel on infarct volume was observed in a clinical trial study (Warach et al., 2006). This is explained by that very low concentrations of glycine are required for activation of the NMDA receptor (Johnson and Ascher, 1987; McNamara and Dingledine, 1990). The NMDAassociated glycine binding site appears to be fully saturated by <1 lmM glycine. Normal level of glycine in the brain interstitial space is about 10 lmM, indicating that the NMDA-associated glycine site would normally be occupied by glycine under physiological conditions (Giuli, 2002). Meanwhile, glycine is a very effective drug for treatment of acute ischemic stroke (Gusev et al., 1999; Zhao et al., 2005). The mechanism is associated with an essential increase in pial vessels diameters when glycine level rises locally (Podoprigora et al., 2005). This mechanism may also contribute to glycine anatagonizing MCAO impair because an equivalent dose of glycine was used in the present study.
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Fig. 5. Protein expression of bcl-2, bax, bcl-2/bax and FasL in the infarct middle cerebral artery region in mice 24 h after MCAO. (A) Representative western blots of bcl-2. (B) Quantitative analysis of bcl-2 (n = 4). (C) Representative western blots of bax. (D) Quantitative analysis of bax (n = 4). (E) Ratio of bcl-2/bax (n = 4). (F) Representative western blots of FasL. (G) Quantitative analysis of FasL (n = 4). Values are the mean ± SE; #P < 0.05 versus sham group; ⁄P < 0.05 versus vehicle group.
Apoptotic neuron also represents an important therapeutic target in the ischemic penumbral area (Ya et al., 2010). Caspase-3 plays a key role in apoptotic cell death and inhibiting caspase-3 cleavage is believed to benefit diminishing cerebral ischemic injury (Li et al., 2010a,b). This is confirmed by our observation that marked increase in TUNEL-staining cells and cleaved caspase-3 in the ischemic mouse penumbra could be effectively dampened by administration of glycine. This is consistent with a report from an in vitro ischemic model that glycine reduced apoptosis in the intermediate area of the ischemic zone (Lobysheva et al., 2009).
As an actively regulated form of cell death, apoptosis is mediated by two pathways: extrinsic pathway and intrinsic pathway. The extrinsic pathway is activated by binding of Fas ligand with Fas, which in turn triggers reactions of procaspases. The intrinsic pathway is activated by diverse stimuli. These signals are transduced to the mitochondria and endoplasmic reticulum by bcl-2 proteins which finally activate downstream procaspases (Whelan et al., 2010). Both apoptotic pathways are the downstream mechanisms by which JNK promotes neuronal cell death (Kuan et al., 2003; Putcha et al., 2003; Okuno et al., 2004; Carboni et al.,
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Fig. 6. Glycine attenuates OGD damage in SH-SY5Y cells. Cells were cultured in a HEPES-buffered glucose-free medium under hypoxic conditions (5% CO2, 95% N2) for 12 h and were replaced in DMEM under normal conditions for another 24 h. Glycine was added to the culture medium before OGD treatment and kept until end of test. Values are the mean ± SE (n = 6); ##P < 0.01 versus control group (con); ⁄ P < 0.05 versus OGD group (OGD).
2005; Soriano et al., 2008; Li et al., 2010a,b). Glycine may inhibit both extrinsic and intrinsic apoptotic pathways in the mouse I/R brain, because it down-regulated both of Fas and FasL and up-regulated bcl-2 and bcl-2/bax ratio, although there was not obvious difference in bax protein level between vehicle and glycine-high
group in this study. Bax is a pro-apoptotic and bcl-2 is an antiapoptotic protein in the bcl-2 family. They serve as distinct regulators of apoptosis at its early stages (Jeong et al., 2008). The 24 h time point may be too early for measurements of bcl-2/bax protein levels in cerebral ischemia, such that the change in vehicle group is minimal and similar to baseline level in sham group (Cheyuo et al., 2011). Ischemia/reperfusion promotes the release of bax from bcl2/bax dimers via JNK phosphorylating bcl-2. This could be dampened by glycine. Whether glycine inhibits directly phosphorylation of bcl-2 needs to be investigated. Our previous study indicated that glycine protects ATP-depleted MDCK cells against cell death (Jiang et al., 2010). OGD reoxygenation, an in vitro model that more closely mimics in vivo cerebral I/R than ATP depletion, was used in this study. Our results confirmed the protective effect of glycine on OGD-insulted SH-SY5Y cell damage. More important, we demonstrated that the glycine’ protective effect was dependent on the expression of GlyR a1 in SH-SY5Y cells. GlyR belongs to the cys-loop family of ligand-gated ion channels. GlyRs are able to form homomeric receptors, composed of a subunits, or heteromeric receptors, composed of a and of b-subunits and in a putative 3b/2a or 2b/3a stoichiometry (Grudzinska et al., 2005). As a ligand of GlyR, glycine is known as a cytoprotector against ischemia or other insults induced damage. The cytoprotective effect of glycine is postulated to be mediated by GlyR, because other ligands of GlyR, such as L-serine and b-alanine, present a cytoprotection similar to glycine (Weinberg et al., 1991; Nagatomi et al., 1997; Wang et al., 2010). However, this hypothesis is challenged by some facts. For example, enhancement of synaptic NMDA receptors by glycine, a co-agonist of NMDA receptor (Danysz and Parsons, 1998), exerts a protective effect against neuronal death (Liu et al., 2007). These facts imply that the NMDA receptor
Fig. 7. Knockdown of glycine receptor on cytoprotection of glycine in SH-SY5Y cells. (A) Transfection of siRNAs on expression of GlyR a1 in SH-SY5Y cells. Expression of glycine receptor a1 was estimated by western blot using an antibody against GlyR a1. (B) Quantitative analysis of GlyR a1 expression (n = 3). (C) Transfection of siRNAs on MTT activity in cells (n = 6). (D) Transfection of siRNAs on LDH release from cells (n = 5). Values are the mean ± SE; ⁄P < 0.05; ⁄⁄P < 0.01.
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Fig. 8. Block of glycine receptor or/and NMDA receptor on cytoprotection of glycine in primary neurons. (A) Blocking of glycine recptor with a glycine receptor antibody (lowdose, 2 lg/ml; high-dose, 10 lg/ml, n = 7 per group) on LDH release from neurons after OGD insult. (B) Representative western blots of cleaved caspase-3. (C) Quantitative analysis of cleaved caspase-3. The band density values were calculated as a ratio of b-actin (n = 3). (D) Blocking NMDA receptor by treatment with MK-801 (MK) or/and an antibody against glycine receptor on LDH release from neurons after OGD insult (n = 7). Values are the mean ± SE; ##P < 0.01 versus control group (con); ⁄⁄P < 0.01 versus OGD group (OGD).
may also be engaged in glycine-induced cytoprotection. Our in vitro experiments clearly showed that knockdown of endogenous GlyR a1 by RNA interference could abolish the protective effects of glycine against OGD insult to SH-SY5Y cells. This result was further confirmed by an observation in primary cultured neurons using a specific antibody to block GlyR and a NMDA receptor antagonist to block NMDA receptor. We found that the cytoprotective effect of glycine could be abolished by pretreatment of cells with an antibody against GlyR. However, the cytoprotective effect of MK-801, a NMDA receptor antagonist, seems to be independent upon glycine or GlyR. Therefore, glycine’s protective effect against I/R injury in neurons could be mediated by GlyR. We demonstrated for the first time that application of glycine effectively inhibits transient brain I/R-induced neuronal apoptosis in vivo. The mechanism may be via suppressing the ‘‘intrinsic’’ (i.e., mitochondria) and the ‘‘extrinsic’’ (i.e., death receptor) apoptotic pathways. Thus, glycine may be useful in the therapy for cerebral I/R injury.
Acknowledgements This work was supported by National Basic Research Program (973) Grant (NO. 2012CB517503 and 2011CB503903) and the project of National Natural Science Foundation of China (NO. 30730044 and 81070120) to Qi Chen, the project of National Natural Science Foundation of China Grant (NO. 81000118) to
Jingjing Ben and the project of National Natural Science Foundation of China Grant (NO. 81100857) to Xiaoyu Li.
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Contents lists available at SciVerse ScienceDirect
Neurochemistry International journal homepage: www.elsevier.com/locate/nci
Glycine attenuates cerebral ischemia/reperfusion injury by inhibiting neuronal apoptosis in mice Yan Lu a,b, Jing Zhang a,b, Bingqing Ma a, Kexue Li a,b, Xiaoyu Li a, Hui Bai b, Qing Yang b, Xudong Zhu a,b, Jingjing Ben a,b, Qi Chen a,b,⇑ a b
Atherosclerosis Research Centre, Laboratory of Molecular Intervention with Cardiovascular Diseases, Nanjing Medical University, Nanjing 210029, PR China State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, PR China
a r t i c l e
i n f o
Article history: Received 5 March 2012 Received in revised form 18 June 2012 Accepted 3 July 2012 Available online 10 July 2012 Keywords: Glycine Cerebral ischemia/reperfusion Apoptosis SH-SY5Y cell siRNA Neurons Glycine receptor
a b s t r a c t Glycine is a cytoprotector to protect cells against ischemic damage by counteracting neuronal depolarization. However, whether it can directly inhibit neuronal apoptosis is unknown. In this study, we demonstrated that glycine could attenuate ischemia/reperfusion (I/R) induced cerebral infarction and improved neurological outcomes in mice. The protective effect of glycine was associated with reduction of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) positive cells, deactivation of phosphor-JNK, inhibition of caspase-3 cleavage, down-regulation of FasL/Fas, and up-regulation of bcl-2 and bcl-2/bax in the mouse I/R penumbra. The beneficial effect of glycine against oxygen and glucose deprivation (OGD) induced injury was also confirmed in SH-SY5Y cells as well as in primary cultured neurons, which was significantly dampened by knockdown of glycine receptor a1 (GlyR a1) with siRNA transfection or by preventing glycine binding with glycine receptor using a specific antibody against glycine receptor. These results suggest that glycine antagonize cerebral I/R induced injury by inhibiting apoptosis in mice. Glycine could block both extrinsic and intrinsic apoptotic pathways for which GlyR may be required. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Stroke is a leading cause of death and adult disability worldwide. Of all strokes, ischemic stroke accounts for approximately 80% (Feigin et al., 2003; Liu et al., 2010). Thrombolysis and revascularization of the obstructed blood vessels are effective and safe therapies to restore the cerebral blood flow. However, the efficacy of these interventions is satisfactory in a selected few types of stroke and is limited by a narrow time window (usually in the very early period) (Hankey and Warlow, 1999; Kwiatkowski et al., 1999). Thus, finding more effective therapeutic neuroprotective agents has become a priority in the field. Abbreviations: CNS, central nervous system; ERK1/2, extracellular signal-regulated kinase 1 and 2; GlyRs, glycine receptors; GlyR a1, glycine receptor a1; IACUC, institutional animal care and use committee; I/R, ischemia/reperfusion; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MCAO, middle cerebral artery occlusion; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; NMDA, N-methyl-D-aspartate; OGD, oxygen and glucose deprivation; TTC, 2,3,5triphenyl tetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. ⇑ Corresponding author. Address: Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, PR China. Tel.: +86 25 86862610; fax: +86 25 86508960. E-mail address: [email protected] (Q. Chen). 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.07.005
Inhibitory amino acid taurine and L-serine can protect the brain from ischemic injury. It is supposed to be mediated by activating glycine receptors (GlyRs) (Wang et al., 2007, 2010). GlyRs, as well as glycine-containing fibers and cell bodies, are widely distributed in brain rather than restricted to the spinal cord and brain stem (Legendre, 2001). As a major inhibitory neurotransmitter, glycine can activate GlyR to reach chloride influx induced postsynaptic hyperpolarization and reduces neuronal excitability. This antiexcitotoxic property is by counteracting neuronal depolarization and subsequent cascade of biochemical events that would result in cell death (Wang et al., 2010). Glycine also exhibits a directive protection against ischemia induced injury to organs and tissues (Yin et al., 2002; Zhang et al., 2003; Omasa et al., 2003; Tang et al., 2006). We showed that cytoprotection against ATP depletion by glycine is mediated by GlyR (Pan et al., 2005). The extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase and AKT pathways constitute GlyR-coupled signaling pathways (Jiang et al., 2010), suggesting a potential anti-apoptotic property should be involved in cytoprotection of glycine. Apoptosis occurs in the delayed neuronal death in the ischemic lesions (Kametsu et al., 2003; Waldmeier, 2003). In order to clarify if glycine antagonizes ischemic injury by an anti-apoptotic mechanism in the brain, we used an experimental transient middle cerebral artery occlusion
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(MCAO) model in mice. Our results demonstrated that glycine may attenuate mouse cerebral ischemia/reperfusion (I/R) injury by inhibiting cell apoptosis. 2. Materials and methods 2.1. Animals Male ICR mice were supplied by the Experimental Animal Center of Nanjing Medical University, Nanjing, China. The experimental protocol was approved by Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University. All experiments were performed in accordance with international standards on the ethical treatment of animals. Mice were housed under climatecontrolled conditions with a 12-h light/dark cycle and provided standard food and water ad libitum. Glycine (Sigma, St. Louis, MO, USA) was dissolved in physiological saline and was intraperitoneally injected into mice 30 min before MCAO for the pretreatment study. All MCAO mice were divided into receiving vehicle, low-dose glycine (250 mg/kg), and high-dose glycine (1000 mg/ kg) groups. For the posttreatment experiments, drug treatments were given via intraperitoneal injection 10 min after reperfusion.
age analyzer. Infarct volume of each slice was obtained by multiplying the infarct area by 2-mm thickness. Total infarct volume was determined by summing up infarct volume of five consecutive slices. All brain slices were analyzed for their infarct volume using the Image-J analysis software. Percentage infarct volume was calculated as follows: [(VC VL)/VC] 100, where VC is the volume of control hemisphere (Right side), VL is the volume of non-infarcted tissue in the lesioned hemisphere (Right side). 2.4. TUNEL staining Samples from sham-operated, vehicle, and glycine-high groups were used for experiments. Mice were deeply anesthetized with chloral hydrate 72 h after reperfusion. After transcardiac perfusion with 50 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4), brains were removed and stored in the same paraformaldehyde solution overnight. Multiple, paraffin-embedded, coronal sections (4 lm thick) were taken from brain and stained by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. The apoptotic neurons were counted using an inverted microscope (Olympus BX51; Japan) in the peri-infarct cortex (three fields were counted in each case at 400 magnification) in a blinded manner.
2.2. MCAO model
2.5. Western blot analysis
Transient focal cerebral ischemia was induced by right MCAO with a modified intralumenal filament technique (Hata et al., 1998). Briefly, mice were anesthetized by 3.5% chloral hydrate (1 mg/kg). A 5-0 monofilament surgical nylon suture with a heatblunted tip was introduced into the left internal carotid artery through the stump of the external carotid to the base of the right to stop blood flow. After 90 min, the filament was withdrawn to allow blood reperfusion. Meanwhile the ipsilateral common carotid artery remained ligated, the neck skin was closed, and the skin was sutured. Rectal temperature, monitored with a digital thermometer inserted 2 cm into the anus, was maintained at 37– 38 °C throughout the operation using a feed back-regulated heating blanket. All animals recovered from anesthesia within 30 min of wound closure. Sham-operated animals underwent the same procedure except for MCAO.
The right ischemic cortex in the middle cerebral artery region (middle cerebral artery cortex) was obtained from preoperation (healthy control) and 24 h after MCAO. Protein concentration was determined using BCA protein assay kit (PIERCE, Rockford, IL, USA). Individual samples (60 lg each) were separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, USA). Immunoblotting was performed by incubation of the membrane in 5% dry milk for 1 h and in the specific antibody overnight at 4 °C. Rabbit antibodies against P-JNK, caspase-3, bcl-2 and bax were obtained from Cell Signaling Techonology (Beverly, MA, USA). Rabbit anti-FasL antibody was obtained from Abcam (Cambridge, UK). Rabbit anti-GlyRa1/2 antibody was obtained from Abd serotec (Oxford, UK). After washing twice with Tris-buffered saline-Tween 20, each membrane was incubated with a secondary antibody (1:5000) for 2 h. Cellular b-actin or GAPDH was used as a loading control to prove that all of the lanes were loaded with the same amount of protein. The membranes were then developed using an enhanced chemiluminescence system. The figures were analyzed with Image J.
2.3. Measurement of the area of early ischemic brain injury Mice were examined for neurological deficit using a four-tiered grading system before giving anesthesia, 24 h, and 72 h after MCAO and reperfusion (Connolly et al., 1996a,b). A score of 1 was given if the animal was demonstrated normal spontaneous movements; 2 was given if the animal was noted to be turning to the right (i.e., clockwise circles) when viewed from above (i.e., toward the contralateral side); 3 was given if the animal was observed to spin longitudinally (clockwise when viewed from the tail); and 4 was given if the animal was crouched on all fours, unresponsive to noxious stimuli. This scoring system is based upon similar scoring systems used in rats (Bederson et al., 1986; Menzies et al., 1992) which are based upon the contralateral movement of animals with stroke. After cerebral infarction, the contralateral side is ‘‘weak’’ and so the animal tends to turn toward the weakened side. Mice were killed by decapitation 24 h or 72 h after MCAO. Brains were removed and chilled in ice-cold saline for 5 min. Four 2-mm consecutive coronal slices were made by using a brain slicer, beginning from the anterior pole. Slices were incubated in saline solution containing 2% 2,3,5-triphenyl tetrazolium chloride (TTC) (Sigma, St. Louis, MO, USA) at 37 °C for 30 min. They were then fixed by 10% formalin neutral buffer solution (pH 7.4) for 1 h. Infarct area in each slice was evaluated by scanned digital images with an im-
2.6. RNA extraction and real-time PCR Total RNA was isolated using Trizol reagent (Invitrogen, San Diego, CA, USA) in accordance with the manufacturer’s protocol. After extraction, 0.5 lg of total RNA was used as template to synthesize cDNA using a first strand synthesis kit (Invitrogen, San Diego, CA, USA). The cDNA from this synthesis was then used in quantitative real-time PCR analysis (ABI-Prism 7700 sequence detection system; Applied Biosystems, Foster City, CA, USA) using SYBR Green dye. The following primer pairs for murine were used: Fasl, 50 TGAATTACCCATGTCCCCAG-30 (forward) and 50 -AAACTGACCCTGGAGGAGCC-30 (reverse); Fas, 50 -TGGCAGAGGAGCCTAGTTGT-30 (forward) and 50 -CACACCCAGGAACAGTCCTT-30 (reverse); bcl-2, 50 -ATGATAACCGGGAGATCGTG-30 (forward) and 50 -GTTCAGGTACTCAGTCACC-30 (reverse); bax, 50 -ACCAGCTCTGAACAGATCATG-30 (forward) and 50 -TGGTCTGGATCCAGACAAG-30 (reverse); actin, 50 TTCGTTGCCGGTCCACA-30 (forward) and 50 -ACCAGCGCAGCGATATCG-30 (reverse) (Pinkoski et al., 2002; Guo et al., 2003; Symonds et al., 2005; Yoshimatsu et al., 2009).
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2.7. Oxygen-glucose deprivation We used oxygen and glucose deprivation (OGD) reoxygenation, an in vitro model that best mimics in vivo cerebral ischemia reperfusion, in the study. Cells were washed once. During OGD, the medium was changed in a HEPES-buffered glucose-free medium (pH 7.4) containing 154 mmol/L NaCl, 5.6 mmol/L KCl, 2.3 mmol/L CaCl2, 1.0 mmol/L MgCl2, 3.6 mmol/L NaHCO3, and 5 mmol/L HEPES. OGD was induced by incubating cells in this medium and placing cells in an anaerobic chamber with an atmosphere of 95% N2 and 5% CO2 at 37 °C (Liu et al., 2010). OGD was terminated after 12 h by replacing the glucose-free medium back to DMEM under normal conditions for another 24 h as an injury reperfusion model in the following experiments. Control cells were maintained normally. Glycine was added to the culture medium before OGD treatment and throughout the OGD reperfusion.
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2 mM glutamine. The medium were replaced with fresh medium every 3 days. Experiments were performed on days 7–10. Neurons were pre-treated for 1 h with 10 lg/ml, 2 lg/ml antibody against GlyR, 10 lg/ml normal rabbit IgG, or pre-treated for 30 min with 10 lM MK-801 before OGD damage. Neurons were subjected to OGD for 3 h followed by 24 h of reoxygenation. Glycine was added to the culture medium before OGD treatment and throughout the OGD reperfusion.
2.12. Statistical analysis All values are presented as mean ± standard error. Data of two groups were analyzed for statistical significance with Student’s ttest. Multiple comparisons were made by using one-way ANOVA. Differences between means were considered significant if p < 0.05.
2.8. siRNA transfection 3. Results Transfections were performed by using Lipofectamine 2000 (Invitrogen, San Diego, CA, USA). The target sequence in human GlyR a1 gene was as follows: GGCCUAUAAUGAAUACCCUtt and AGGGUAUUCAUUAUAGGCCag. A scrambled siRNA was used as a negative control. All oligonucleotides were obtained from Ambion RNA. SH-SY5Y cells were seeded 1 day prior to transfection. They were 30% confluent when they were transfected with 25 nM positive or scrambled oligonucleotides in Lipofectamine 2000 and Opti-MEM (Invitrogen, San Diego, CA, USA) without serum for 72 h (Zhu et al., 2011). Expression of glycine receptor a1 was estimated by western blot using an antibody against GlyR a1. 2.9. Measurements of cell damage Cellular 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed by addition of 25 ll MTT stock solution (5 mg/ml) to each well. After incubation for 4 h, the MTT solution was carefully decanted off and formazan was dissolved with 200 ll DMSO. The absorbance was measured at 492 nm using a 96-well plate ELISA reader (CliniBio 128, Austria).
3.1. Glycine attenuates cerebral I/R injury in mice To test the effect of glycine on cerebral ischemia in mice, infarct volume and neurological score were recorded both 24 and 72 h after MCAO injury. The neurological score before reperfusion was not significantly different among the groups (data not shown). However, the infarct volume was significantly diminished (Fig. 1A and B) and the neurological score was improved by glycine administration 24 h after MCAO (Fig. 1C). The neuroprotective effects of glycine were also found in mice 72 h post MCAO injury (Fig. 1D–F). Treatment with a high dose of glycine reduced significantly total infarct volume comparing with the vehicle group both 24 h (48.86 ± 3.9 mm3 versus 64.66 ± 2.58 mm3, Fig. 1B) and 72 h (49.02 ± 2.2 mm3 versus 65.42 ± 3.4 mm3, Fig. 1E) after MCAO. Consistently, a significant improvement in neurological score was observed in glycine-high groups comparing with vehicle groups both 24 h (2.59 ± 0.54 versus 1.86 ± 0.23, Fig. 1C) and 72 h (2.88 ± 0.79 versus 2 ± 0.5, Fig. 1F) after MCAO. These results suggest that glycine may attenuate cerebral I/R injury in mice.
2.10. LDH measurement
3.2. Glycine inhibits I/R-induced neuronal apoptosis in mice
Cell injury was determined by measuring the amount of lactate dehydrogenase (LDH) released into the medium (Koh and Choi, 1987). Cultures were exposed to OGD and treated with glycine for 12 h followed by 24 h reoxygenation. The medium was collected before cell was permeabilized with equal volume of 0.4% (v/v) Triton. The activities of extracellular and intracellular LDH were measured separately. The extracellular LDH activity was divided by the total LDH activity (extracellular activity plus intracellular activity) to calculate the percentage of LDH release.
To determine if the neuroprotective effect of glycine is by inhibiting apoptosis, we analyzed penumbra apoptotic status with TUNEL staining. No TUNEL-positive neurons were detected in the sham group (Fig. 2A) but they were evident in vehicle group 72 h after MCAO. Number of TUNEL-positive neurons was significantly decreased by treatment with glycine (Fig. 2B). Furthermore, we checked expression levels of phosphorylated c-Jun N-terminal kinase (JNK) and cleavaged caspase-3 in the middle cerebral artery cortex by western blot. As shown in Fig. 3A and B, MCAO activated JNK phosphorylation 24 h post-MCAO. Glycine treatment could dampen it significantly. Effect of high dose of glycine was much stronger than that of low dose of glycine. Level of phosphorylated JNK in glycine-high group was even lower than that of sham group but was not statistically significant (sham: 127.79 ± 11.7%; vehicle: 170.01 ± 7.2%; glycine: 135.42 ± 8.9%; glycine-high: 89.26 ± 19.1%, Fig. 3B). The cleaved caspase-3 expression 72 h post-MCAO (Fig. 3C and D) was significantly increased in the vehicle groups. They were lower in glycine and glycine-high groups compared with that of vehicle group. Difference between vehicle and glycine-high group was statistically significant (sham: 38.05 ± 5.4%; vehicle: 77.78 ± 8.5%; glycine: 55.9 ± 3.6%; glycinehigh: 33.07 ± 11.2%, Fig. 3D).
2.11. Neuronal culture and treatment Neurons were isolated as previously described (Ming et al., 2006). Briefly, cerebral cortices were dissected from postnatal day 0 ICR mice and incubated in 0.125% trypsin for 30 min at 37 °C. Tissues were then triturated with fire-polished glass pipettes and plated on poly-L-lysine-coated plates or dishes at a density of 1 105. Neurons were cultured with DMEM/F12 (Invitrogen, San Diego, CA, USA) (1:1) supplemented with 10% fetal bovine serum. Cells were maintained in incubators at 37 °C under 5% CO2 atmosphere. After 24 h, the culture medium was changed to neurobasal medium supplemented with 2% B27 (both by Gibco, NY, USA) and
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Fig. 1. Neuroprotective effect of glycine against cerebral ischemic/reperfusion injury in mice. Mice were treated with glycine (low-dose, 250 mg/kg, n = 10; high-dose, 1000 mg/kg, n = 10) or vehicle (n = 8). Sham-operated animals (sham, n = 5) underwent the same procedure except for MCAO. Mice were examined for neurological deficit or killed to measure the infarct volume after MCAO. (A) Representative photos of the mouse brain 24 h after MCAO. (B) Quantitative analysis of total infarct volume in mice 24 h after MCAO. (C) Neurological score 24 h after MCAO. (D) Representative photos of the mouse brain 72 h after MCAO. (E) Quantitative analysis of total infarct volume in mice 72 h after MCAO. (F) Neurological score 72 h after MCAO. Values are shown as the mean ± SE; ##P < 0.01 versus sham group; ⁄P < 0.05 versus vehicle group, ⁄⁄P < 0.01 versus vehicle group.
3.3. Glycine up-regulates bcl-2 and bcl-2/bax and down-regulates FasL/Fas after transient MCAO in mice
3.4. Knockdown of glycine receptor diminishes glycine protection against OGD injury in SH-SY5Y cells
Having established that glycine can markedly inhibit JNK phosphorylation and caspase-3 cleavage in the I/R mouse brain, we wondered which apoptotic pathways were targeted by glycine in vivo. Bcl-2/bax is a marker of intrinsic apoptotic pathway and FasL/Fas is an extrinsic apoptotic pathway marker. We found that bcl-2/bax mRNA ratio was decreased in the focal brain after I/R injury. Glycine treatment resulted in a significant increase in bcl-2/ bax compared with vehicle group (Fig. 4A). In accordance with it, western blot analysis confirmed that there was no significant difference in levels of bcl-2, bax, and bcl-2/bax between sham and vehicle group 24 h after MCAO. Dramatic increases in bcl-2 (sham: 106.89 ± 18.6%; vehicle: 115.41 ± 7.9%; glycine-high: 174.81 ± 20%, Fig. 5B) and bcl-2/bax proteins (sham: 0.88 ± 0.1; vehicle: 1.03 ± 0.1; glycine-high: 1.5 ± 0.2, Fig. 5E) were found when glycine treatment was performed. But glycine did not influence bax expression significantly (Fig. 5C and D). We also found that both mRNA and protein levels of FasL in the infarct middle cerebral artery region were significantly increased after treatment with glycine but not with saline (Figs. 4B and 5F and G). Coincidentally, Fas mRNA level was also significantly increased after I/R which was dampened by treatment with glycine in mice (Fig. 4C). These data indicated that glycine may target at both bcl-2/bax and FasL/Fas mediated apoptosis to attenuate cerebral I/R injury in mice.
To further investigate mechanism underlying glycine’s neuroprotection, we used an OGD cell model in vitro. MTT activity in SH-SY5Y cells was significantly decreased after 12 h OGD followed by 24 h reperfusion. As shown in Fig. 6, treatment with glycine could antagonize the OGD injury in cells obviously. It is dosedependent from 1 to 20 mM. We used 5 mM as a working dose for glycine treatment in vitro in the following experiments. Glycine is a ligand of GlyR. To clarify if GlyR is requisite for neuroprotection of glycine, GlyR knockdown was performed by RNA interference. When GlyR a1 protein expression was knocked down by 70% in SH-SY5Y cells, 71% of recovering MTT effect by glycine was abolished in OGD cells (Fig. 7A–C). Meanwhile, knockdown of GlyR a1 led to a significant increase in LDH release (by 60%) from cells even in the presence of glycine (Fig. 7D). These data revealed that GlyR a1 may be requisite for cytoprotection of glycine against OGD injury in SH-SY5Y cells. 3.5. Glycine increases cell viability and prevents cell death induced by OGD in primary cultured neurons We also observed cytoprotective effect of glycine in primary cultured neurons. The release of LDH from cells occurs with the loss of plasma membrane integrity, a process most often associated with necrotic cell death. Fig. 8A showed that OGD treatment re-
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Fig. 2. TUNEL-staining cells 72 h after MCAO in mice. (A) Representative photomicrographs of TUNEL-positive cells in ischemic penumbra. (B) Quantitative analysis of TUNEL-positive neurons in ischemic penumbra. Values are the mean ± SE (n = 5); ##P < 0.01 versus sham group; ⁄⁄P < 0.01 versus vehicle group.
Fig. 3. JNK phosphorylation and cleaved caspase-3 in the infarct middle cerebral artery region in mice. The left ischemic middle cerebral artery cortex in mice was obtained 24 h after MCAO for measurement of JNK phosphorylation and 72 h after MCAO for measurement of cleaved caspase-3 separately (n = 4, respectively). (A) Representative western blots of phospho-JNK. (B) Quantitative analysis of JNK phosphorylation. (C) Representative western blots of cleaved caspase-3. (D) Quantitative analysis of cleaved caspase-3. The band density values were calculated as a ratio of b-actin. Values are the mean ± SE; #P < 0.05 versus sham group; ⁄P < 0.05 versus vehicle group.
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Fig. 4. mRNA levels of bcl-2/bax, FasL, and Fas in the infarct middle cerebral artery region in mice 24 h after MCAO. (A) Ratio of bcl-2/bax mRNA levels (n = 5). (B) FasL mRNA levels (n = 5). (C) Fas mRNA levels (n = 5). Values are the mean ± SE; #P < 0.05 versus sham group; ##P < 0.01 versus sham group; ⁄⁄P < 0.01 versus vehicle group.
sulted in a significant increase in LDH level compared with control. Glycine 5 mM blocked OGD-induced LDH release by 50%, which was inhibited by pre-treated with an antibody against GlyR. Furthermore, we checked expression levels of cleaved caspase-3 in OGD-insulted neurons. As shown in Fig. 8B and C, OGD treatment led to an increase in cleaved caspase-3 which was dampened in the presence of glycine. Addition of an antibody against GlyR mostly abolished the neuroprotection of glycine (control: 41.21 ± 11.2%; OGD: 253.02 ± 25.4%; glycine: 112.63 ± 8.9%; antibody: 253.22 ± 45.1%; antibody + glycine: 247.4 ± 30%, Fig. 8C). As a co-agonist of glutamate N-methyl-D-aspartate (NMDA) receptor, the neuroprotection of glycine has been thought to be mediated by NMDA receptor (Liu et al., 2007). We used MK-801, a NMDA receptor antagonist to test the role of NMDA receptor in cytoprotection. Fig. 8D showed that treatment with MK-801 alone provided a significant 60% reduction of LDH release compared with OGD. This effect was increased by simultaneous treatment with glycine but there was not a statistical significance between these two groups. The antibody against glycine receptor did not influence the LDH release in MK-801 and MK-801 plus glycine groups, suggesting that GlyR should not be involved in the MK-801’s effect. 4. Discussion Glycine is one of the main components mediating fast inhibitory neurotransmission in the central nervous system (CNS), in which the strychnine-sensitive GlyR, a ligand-gated anionic channel, is primarily involved in it. Besides this neurotransmitter function, glycine has been shown to antagonize shock and ischemia/reperfusion injury in many other non-neuronal tissues and organs including liver, kidney, heart, intestine, and skeletal muscle
(Gundersen et al., 2005; Qi et al., 2007). The protective mechanism of glycine seems vary in cell types. In renal cells, hepatocytes, and endothelial cells, glycine exerts its cytoprotective effect by stabilizing porous defects in the ischemic plasma membranes (den Eynden et al., 2009). In lung ischemia-reperfusion injury, glycine attenuates I/R injury by reducing oxidative damage and suppressing apoptosis (Omasa et al., 2003). In hepatic ischemia, glycine can also prevent apoptosis-related cell death (Duenschede et al., 2006). We demonstrated that glycine could antagonize cerebral I/R injury by suppressing neuronal apoptosis in mice. As a co-agonist of glutamate N-methyl-D-aspartate (NMDA) receptor, glycine can induce neurotoxicity in organotypic hippocampal slice cultures (Sua´rez et al., 2005). Accordingly, compounds blocking the glycine site on the NMDA receptor have been widely investigated. Gavestinel is a one of the selective antagonists for the glycine site on the NMDA receptor. However, no effect of gavestinel on infarct volume was observed in a clinical trial study (Warach et al., 2006). This is explained by that very low concentrations of glycine are required for activation of the NMDA receptor (Johnson and Ascher, 1987; McNamara and Dingledine, 1990). The NMDAassociated glycine binding site appears to be fully saturated by <1 lmM glycine. Normal level of glycine in the brain interstitial space is about 10 lmM, indicating that the NMDA-associated glycine site would normally be occupied by glycine under physiological conditions (Giuli, 2002). Meanwhile, glycine is a very effective drug for treatment of acute ischemic stroke (Gusev et al., 1999; Zhao et al., 2005). The mechanism is associated with an essential increase in pial vessels diameters when glycine level rises locally (Podoprigora et al., 2005). This mechanism may also contribute to glycine anatagonizing MCAO impair because an equivalent dose of glycine was used in the present study.
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Fig. 5. Protein expression of bcl-2, bax, bcl-2/bax and FasL in the infarct middle cerebral artery region in mice 24 h after MCAO. (A) Representative western blots of bcl-2. (B) Quantitative analysis of bcl-2 (n = 4). (C) Representative western blots of bax. (D) Quantitative analysis of bax (n = 4). (E) Ratio of bcl-2/bax (n = 4). (F) Representative western blots of FasL. (G) Quantitative analysis of FasL (n = 4). Values are the mean ± SE; #P < 0.05 versus sham group; ⁄P < 0.05 versus vehicle group.
Apoptotic neuron also represents an important therapeutic target in the ischemic penumbral area (Ya et al., 2010). Caspase-3 plays a key role in apoptotic cell death and inhibiting caspase-3 cleavage is believed to benefit diminishing cerebral ischemic injury (Li et al., 2010a,b). This is confirmed by our observation that marked increase in TUNEL-staining cells and cleaved caspase-3 in the ischemic mouse penumbra could be effectively dampened by administration of glycine. This is consistent with a report from an in vitro ischemic model that glycine reduced apoptosis in the intermediate area of the ischemic zone (Lobysheva et al., 2009).
As an actively regulated form of cell death, apoptosis is mediated by two pathways: extrinsic pathway and intrinsic pathway. The extrinsic pathway is activated by binding of Fas ligand with Fas, which in turn triggers reactions of procaspases. The intrinsic pathway is activated by diverse stimuli. These signals are transduced to the mitochondria and endoplasmic reticulum by bcl-2 proteins which finally activate downstream procaspases (Whelan et al., 2010). Both apoptotic pathways are the downstream mechanisms by which JNK promotes neuronal cell death (Kuan et al., 2003; Putcha et al., 2003; Okuno et al., 2004; Carboni et al.,
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Fig. 6. Glycine attenuates OGD damage in SH-SY5Y cells. Cells were cultured in a HEPES-buffered glucose-free medium under hypoxic conditions (5% CO2, 95% N2) for 12 h and were replaced in DMEM under normal conditions for another 24 h. Glycine was added to the culture medium before OGD treatment and kept until end of test. Values are the mean ± SE (n = 6); ##P < 0.01 versus control group (con); ⁄ P < 0.05 versus OGD group (OGD).
2005; Soriano et al., 2008; Li et al., 2010a,b). Glycine may inhibit both extrinsic and intrinsic apoptotic pathways in the mouse I/R brain, because it down-regulated both of Fas and FasL and up-regulated bcl-2 and bcl-2/bax ratio, although there was not obvious difference in bax protein level between vehicle and glycine-high
group in this study. Bax is a pro-apoptotic and bcl-2 is an antiapoptotic protein in the bcl-2 family. They serve as distinct regulators of apoptosis at its early stages (Jeong et al., 2008). The 24 h time point may be too early for measurements of bcl-2/bax protein levels in cerebral ischemia, such that the change in vehicle group is minimal and similar to baseline level in sham group (Cheyuo et al., 2011). Ischemia/reperfusion promotes the release of bax from bcl2/bax dimers via JNK phosphorylating bcl-2. This could be dampened by glycine. Whether glycine inhibits directly phosphorylation of bcl-2 needs to be investigated. Our previous study indicated that glycine protects ATP-depleted MDCK cells against cell death (Jiang et al., 2010). OGD reoxygenation, an in vitro model that more closely mimics in vivo cerebral I/R than ATP depletion, was used in this study. Our results confirmed the protective effect of glycine on OGD-insulted SH-SY5Y cell damage. More important, we demonstrated that the glycine’ protective effect was dependent on the expression of GlyR a1 in SH-SY5Y cells. GlyR belongs to the cys-loop family of ligand-gated ion channels. GlyRs are able to form homomeric receptors, composed of a subunits, or heteromeric receptors, composed of a and of b-subunits and in a putative 3b/2a or 2b/3a stoichiometry (Grudzinska et al., 2005). As a ligand of GlyR, glycine is known as a cytoprotector against ischemia or other insults induced damage. The cytoprotective effect of glycine is postulated to be mediated by GlyR, because other ligands of GlyR, such as L-serine and b-alanine, present a cytoprotection similar to glycine (Weinberg et al., 1991; Nagatomi et al., 1997; Wang et al., 2010). However, this hypothesis is challenged by some facts. For example, enhancement of synaptic NMDA receptors by glycine, a co-agonist of NMDA receptor (Danysz and Parsons, 1998), exerts a protective effect against neuronal death (Liu et al., 2007). These facts imply that the NMDA receptor
Fig. 7. Knockdown of glycine receptor on cytoprotection of glycine in SH-SY5Y cells. (A) Transfection of siRNAs on expression of GlyR a1 in SH-SY5Y cells. Expression of glycine receptor a1 was estimated by western blot using an antibody against GlyR a1. (B) Quantitative analysis of GlyR a1 expression (n = 3). (C) Transfection of siRNAs on MTT activity in cells (n = 6). (D) Transfection of siRNAs on LDH release from cells (n = 5). Values are the mean ± SE; ⁄P < 0.05; ⁄⁄P < 0.01.
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Fig. 8. Block of glycine receptor or/and NMDA receptor on cytoprotection of glycine in primary neurons. (A) Blocking of glycine recptor with a glycine receptor antibody (lowdose, 2 lg/ml; high-dose, 10 lg/ml, n = 7 per group) on LDH release from neurons after OGD insult. (B) Representative western blots of cleaved caspase-3. (C) Quantitative analysis of cleaved caspase-3. The band density values were calculated as a ratio of b-actin (n = 3). (D) Blocking NMDA receptor by treatment with MK-801 (MK) or/and an antibody against glycine receptor on LDH release from neurons after OGD insult (n = 7). Values are the mean ± SE; ##P < 0.01 versus control group (con); ⁄⁄P < 0.01 versus OGD group (OGD).
may also be engaged in glycine-induced cytoprotection. Our in vitro experiments clearly showed that knockdown of endogenous GlyR a1 by RNA interference could abolish the protective effects of glycine against OGD insult to SH-SY5Y cells. This result was further confirmed by an observation in primary cultured neurons using a specific antibody to block GlyR and a NMDA receptor antagonist to block NMDA receptor. We found that the cytoprotective effect of glycine could be abolished by pretreatment of cells with an antibody against GlyR. However, the cytoprotective effect of MK-801, a NMDA receptor antagonist, seems to be independent upon glycine or GlyR. Therefore, glycine’s protective effect against I/R injury in neurons could be mediated by GlyR. We demonstrated for the first time that application of glycine effectively inhibits transient brain I/R-induced neuronal apoptosis in vivo. The mechanism may be via suppressing the ‘‘intrinsic’’ (i.e., mitochondria) and the ‘‘extrinsic’’ (i.e., death receptor) apoptotic pathways. Thus, glycine may be useful in the therapy for cerebral I/R injury.
Acknowledgements This work was supported by National Basic Research Program (973) Grant (NO. 2012CB517503 and 2011CB503903) and the project of National Natural Science Foundation of China (NO. 30730044 and 81070120) to Qi Chen, the project of National Natural Science Foundation of China Grant (NO. 81000118) to
Jingjing Ben and the project of National Natural Science Foundation of China Grant (NO. 81100857) to Xiaoyu Li.
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