Neurochem Res DOI 10.1007/s11064-015-1580-7
ORIGINAL PAPER
Parecoxib Protects Mouse Cortical Neurons Against OGD/R Induced Neurotoxicity by Up-Regulating Bcl-2 Yueling Wang1 • Wenjuan Ma1 • Aijun Jia2 • Qulian Guo1
Received: 2 January 2015 / Revised: 6 April 2015 / Accepted: 9 April 2015 Ó Springer Science+Business Media New York 2015
Abstract Ischemic stroke remains a significant problem that is the major cause of death and disability worldwide. Parecoxib is clinically used for short-term management of postoperative pain. Administration of parecoxib in rats has been reported to protect against the cerebral ischemia/ reperfusion. However, the neuroprotective mechanism of parecoxib is still largely unknown. In this study, we found parecoxib could protect against neurotoxicity induced by 4 h oxygen–glucose deprivation (OGD) plus reoxgenation for 20 h, a widely used in vitro model of ischemia/reperfusion. In addition, we characterized the molecular mechanism of parecoxib’s neuroprotection. We found parecoxib was able to activate CREB, and subsequently maintained the expression of Bcl-2, which is an important mitochondria-associated protein. Inhibition of endogenous Bcl-2 expression by transfection of Bcl-2-shRNA significantly attenuated the neuroprotective effects of parecoxib treatment. Furthermore, ATP production assay and mitochondrial membrane potential (DWm) assay suggested that parecoxib exerted neuroprotective effect against OGD/ R by maintaining the function of mitochondria. These data suggested that parecoxib treatment is a potential therapeutic approach for protecting against ischemia/reperfusion injury.
& Qulian Guo [email protected] 1
Department of Anesthesiology, Xiangya Hospital of Central South University, 87 Xiangya Road, Changsha City 410008, Hunan Province, China
2
Department of Respiratory Medicine, The Second Xiangya Hospital, Central South University, Changsha City, Hunan Province, China
Keywords Parecoxib Oxygen–glucose deprivation/ reoxgenation Neurotoxicity Bcl-2 Abbreviations OGD/R Oxygen–glucose deprivation/reoxgenation CREB cAMP-response element binding protein COX-2 Cyclooxygenase-2 LDH Lactate dehydrogenase MACO Middle cerebral artery occlusion
Introduction Ischemic stroke is the common cause of mortality and disability worldwide. Although great efforts have been made in the past decades, the therapeutic methods for patients with ischemic stroke remain limited [1]. Until now, only one pharmacological agent, named recombinant tissue plasminogen activator (rtPA), has been approved by US Food and Drug Administration (FDA) for acute ischemic stroke by breaking down the occluding clot in cerebral vessels and restoring blood flow to the ischemic brain [2]. However, cerebral ischemia/reperfusion can often result in inflammation by upregulation of proinflammatory cytokines including interleukin-1b (IL-1b) and tumor necrosis factora (TNF-a) and cause secondary damage [3]. Therefore, the anti-inflammatory agents are potentially useful for protecting against the cerebral ischemia/reperfusion. Cyclooxygenase-2 (COX-2) is an enzyme that mediates inflammatory process through protaglandins release and inhibition of COX-2 thereby relieves inflammation and pain [4]. Parecoxib, a novel potent and selective COX-2 inhibitor, has been widely used for short-term management of postoperative pain in clinical parctice. Clinical trials
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have indicated that parecoxib is effective and well tolerated for treatment of postoperative pain after dental surgery, orthopedic surgery [5]. Due to the anti-inflammatory property, the neuroprotective effect of parecoxib had been studied previously. An earlier study reported that parecoxib is neuroprotective in spontaneously hypertensive rats after transient middle cerebral artery occlusion, which is closely related to the inflammation inhibition [6]. Another study found that pre-treatment with intravenous parecoxib protects against focal cerebral ischemia/reperfusion injury, and the neuroprotective effects is probably associated with the inhibition of inflammatory reaction [7]. Moreover, our previous study suggested that parecoxib plays neuroprotective role by attenuating postischemic neuronal apoptosis, which mediated by activation of Akt and GSK-3b signaling pathway [8]. However, the underlying neuroprotective mechanisms of parecoxib against cerebral ischemia/reperfusion injury still remain largely unknown. Oxygen–glucose deprivation (OGD) is a classical in vitro model of ischemia, which has been frequently used for mimicking the interruption of the blood supply including oxygen and nutrients to the brain using primary neuronal cultures [9]. In this study, we aimed to investigate the role of parecoxib in the protection against cerebral ischemia/reperfusion injury using OGD plus reoxygenation (OGD/R) model. We found that parecoxib treatment was capable of protecting against OGD/Rinduced neuronal injury in mouse primary cortical neurons. Moreover, we found parecoxib could induce the activation of CREB and the expression of Bcl-2, which is responsible for the neuroprotective effect of parecoxib. Importantly, Bcl-2 protein had been demonstrated to exert neuroprotective effect by maintaining the function of mitochondria [10], we thus examined the role of Bcl-2 in parecoxib’s neuroprotection against OGD/R-induced mitochondria injury. Our results suggested that the neuroprotective effect of parecoxib was closely associated with the functional recovery of mitochondria. The results of present study suggested that parecoxib treatment is potentially a therapeutic approach for intervention of ischemia/ reperfusion injury.
Materials and Methods All animal procedures were carried out following protocols approved by the Ethic Committee of the Xiangya Hosipital of Central South University.
Materials Dulbecco’s modification of Eagle’s medium (DMEM), Neurobasal medium (NBM), glutamine, B27 supplements, LipofectamineTM 2000 were purchased from Invitrogen.
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Fetal bovine serum and 0.05 % trypsin–EDTA were purchased from Gibco. Antibodies against Akt, phosphoSer473-Akt (p-Akt), CREB, phospho-Ser133-CREB (pCREB), caspase 3, cleaved caspase 3 and Bcl-2 were purchased from Cell Signaling Technology. Antibodies against NeuN and GFAP were purchased from Abcam. bactin antibody was obtained from Sigma-Aldrich. Primary Mouse Cortical Neuron Culture Primary mouse cortical neuron cultures were established from C57 BL/6 mouse embryos using methods described before [11]. Briefly, primary mouse cortical neurons were isolated from 15 day mouse embryonic cortex. Dissociated neurons were seeded into 6 or 24-well plates, and maintained in NBM containing 0.3 mM glutamine and 3 % B27 supplements. The NBM was half changed with fresh NBM each 3–4 days. Bcl-2 Knockdown Using Bcl-2-shRNA Bcl-2-shRNA and scrambled shRNA (control shRNA) were inserted in pGFP-V-RS retroviral vector, respectively. The vectors were then transfected into primary cortical neurons according to the manufacturer’s instruction (OriGene). Briefly, for 3 9 105 cells, 1 lg of Bcl-2-shRNA or control shRNA vector was pre-mixed with 3 lL of TurboFectin solution for 30 min, and the mixture was added into NBM of neurons for incubation in a 5 % CO2 incubator for another 48 h. Oxygen–Glucose Deprivation (OGD) Plus Reoxygenation The NBM of days 8 of primary neuron cultures was replaced by serum free DMEM, and the cells were then put in a modular chamber (Billups–Rothenber) perfused with 90 % N2/5 % CO2/5 % H2 for 4 h at 37°C. Following OGD, cells were transferred into the incubator for reoxygenation for 4 h, then treated with parecoxib and followed by reoxygenation for another 16 h (The total reoxygenation time is 20 h). The control group were underwent media change, but were not exposed to OGD and reoxygenation. Lactate Dehydrogenase (LDH) Assay After 4 h OGD plus 20 h reoxygenation, LDH assay was performed to measure neurotoxicity using the LDH detection kit according to the manufacturer’s instruction (Roche). The LDH activity in the untreated normal cell culture medium was detected as ‘‘low control’’, and the LDH activity released from the untreated control neurons was determined as ‘‘high control’’ when the neurons were
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treated with 0.5 % Triton X-100. To determine the percentage of neurotoxicity, the resulting values are substituted in the following equation: Neurotoxicity (%) = (experimental value - low control)/(high control - low control) 9 100.
with 5 lM JC-1 for 20 min at 37°C in the incubator. The flurescent signals of DWm with excitation 545 nm and emission 620 nm were read with the microplate reader (Lifecare Medical). Statistical Analysis
MTT Assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to detect cell viability. Briefly, cells were treated with MTT which was dissolved in DMEM at a final concentration of 1 mg/ml, and incubated at 37°C for 4 h. Supernatant was removed and replaced with 100 ll of DMSO. The optical density (OD) was recorded on the microplate reader (Lifecare Medical) at 570 nm. Cell viability was expressed as a percentage of the control value.
All data were expressed as mean ± SEM. Statistical significance was determined by a one-way ANOVA followed by Tukey–Kramer tests. P value \0.05 was considered significant. At least three independent experiments were performed.
Results Parecoxib Protects Against OGD/R-Induced Neuronal Cell Death
Western Blot Cells were lysed using cell lysis buffer (Cell Signaling Technology). Equal amounts of proteins from neurons were subjected to 4–12 % SDS-PAGE gel and transferred to PVDF membranes. The blot was blocked with 5 % nonfat dried milk, incubated overnight at 4°C with mouse primary antibody. Then, incubation was followed by horseradish peroxidase-conjugated anti-mouse secondary antibodies. Immunolabeling was performed using enhanced chemiluminescence (Amersham Pharmacia Biotech) following manufacturer’s instruction, and then exposed to film (Eastman Kodak Co.). ATP Production Assay ATP production assay was performed to determine the intracellular ATP levels in mouse primary cortical neurons using ATP Bioluminescence assay kit according to manufacturer’s instructions (Roche). After treatment, neurons were lysed with 50 lL cell lysis buffer and shaked for 2 min to ensure completely release of ATP. 5 lL cell lysates were used for protein concentration measurement, the rest of the cell lysates were added with 100 lL luciferase reagent. Then the luminescence was measured by VeritasTM Microplate Luminometer (Turner BioSystems).
To investigate the effect of parecoxib on OGD/R-induced neuronal cell death in cultured primary cortical neurons, the MTT assay was used. Consistent with previous study [12], OGD4 h/R20 h induced a striking increase in cell death, whereas treatment with parecoxib at 4 h of reoxgenation after OGD 4 h resulted in a significantly improvement in the survival rate of neurons, in a dosedependent manner (Fig. 1a). Similarly, in LDH assay, OGD/R largely increased LDH release, but treatment with parecoxib significantly decreased LDH release, thereby indicating that parecoxib can attenuate OGD/R-induced neuronal damage (Fig. 1b). Parecoxib Attenuated OGD/R-Induced Cleavage of Caspase-3 Caspase-3 is a pro-apoptotic protein that is usually used as the maker for cell apoptosis [13, 14]. To investigate the effect of parecoxib on the OGD/R-induced activation of caspase-3, western blot was performed. The results showed that OGD/R significantly increased the cleavage of caspase-3, whereas treatment with parecoxib significantly decreased OGD/R-induced the expression of cleaved caspase-3 when compared with control group, indicating that parecoxib was capable of inhibiting OGD/R-induced neuronal apoptosis (Fig. 2).
Mitochondrial Membrane Potential Mitochondrial membrane potential (DWm) was measured using JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide) mitochondrial membrane potential assay kit according to the manufacturer’s instructions (Cell Technology). After 4 h OGD plus reoxygenation for 20 h, the culture neurons were incubated
Parecoxib Significantly Increases CREB Phosphorylation and Bcl-2 Expression Our previous study indicated that parecoxib was able to increase the expression of Akt phosphorylation (p-Akt) [8], and Akt/CREB signaling played an important role in protecting against ischemia/reperfusion injury [15], the effect
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Fig. 1 Effect of dose-dependent of parecoxib on OGD induced neurotoxicity in mouse primary cortical neurons. a Cell viability was measured by MTT assay at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment alone, #P \ 0.05 compared with PBS plus OGD4 h/
R20 h. b Neurotoxicity was measured by LDH assay at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment alone, #P \ 0.05 compared with PBS plus OGD4 h/R20 h
Fig. 2 Effect of parecoxib on OGD/R induced the cleavage of Caspase-3. Representative Western blot showed that parecoxib inhibited OGD/R induced expression of cleaved Caspase-3. b-actin served as an equal loading control. Quantitative analysis of cleaved
Caspase-3 (fold of control). Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with Control (PBS), #P \ 0.05 compared with parecoxib (0 lM) plus OGD4 h/R20 h
of parecoxib on the p-Akt, p-CREB and a CREB target Bcl-2 protein were detected. As expected, the results showed that parecoxib significantly increased the expression levels of p-Akt, p-CREB and Bcl-2 in a dose-dependent manner (Fig. 3).
shown, OGD/R resulted in the downregulation of CREB phosphorylation and Bcl-2 expression, whereas treatment of parecoxib significantly rescued the inhibitory effect of OGD/R on CREB phosphorylation and Bcl-2 expression (Fig. 4). Therefore, these data implied that Bcl-2 is of significant importance for the neuroprotection role of parecoxib against OGD/R.
Parecoxib Partially Rescued the Inhibitory Effect of OGD/R on CREB Phosphorylation and Bcl-2 Expression OGD/R had been demonstrated to inhibit CREB phosphorylation and Bcl-2 expression [16]. To investigate the role of parecoxib in OGD/R mediated inhibition of CREB phosphorylation and Bcl-2 expression, mouse primary cortical neurons were suffered from OGD for 4 h, and incubated with or without parecoxib at 4 h of reoxygenation, followed by reoxgenation for another 16 h. As previously
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Parecoxib Inhibited OGD/R-Induced Neuronal Injury by Upregulating Bcl-2 Expression To further investigate the effect of Bcl-2 knockdown on the neuroprotective role of parecoxib, Bcl-2-shRNA was transfected into primary cortical neurons. Western blot showed that transfection of Bcl-2-shRNA, but not controlshRNA, led to markedly reduction of Bcl-2 protein expression in primary cortical neurons (Fig. 5a). In LDH
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Fig. 3 Effect of dose-dependent of parecoxib on the expression of p-Akt, p-CREB and Bcl-2. a Representative Western blot showed that dose-dependent of parecoxib induced the expression of p-Akt, p-CREB and Bcl-2. b-Actin served as an equal loading control. b Quantitative analysis of p-Akt (fold of control). Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with Parecoxib
(0 lM). c Quantitative analysis of p-CREB (fold of control). Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with Parecoxib (0 lM). d Quantitative analysis of Bcl-2 (fold of control). Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with Parecoxib (0 lM)
assay, we found that inhibition of endogenous Bcl-2 expression by specific Bcl-2 shRNA in primary cortical neurons significantly attenuated the role of parecoxib against OGD/R-induced neurotoxicity (Fig. 5b). Similar results were also found in MTT assay, transfection with Bcl-2-shRNA significantly inhibited the neuroprotective effect of parecoxib (Fig. 5c). Therefore, these results suggested that parecoxib inhibited OGD/R-induced neuronal injury at least in part through a mechanism involving increased Bcl-2 expression.
improvement of mitochondrial function, mitochondrial membrane potential (DWm) was measured using JC-1 reagents. As expected, DWm value was largely decreased by OGD/R compared to normal neurons, while the reduction was significantly rescued by parecoxib (Fig. 6a). In addition, intracellular ATP levels in neurons were also detected, as it’s another functional marker of mitochondria. Our results showed that intracellular ATP levels in neurons were significantly decreased by OGD/R, but parecoxib treatment led to significant improvement of ATP levels (Fig. 6b). Taken together, these data suggested that parecoxib could inhibit OGD/R-induced mitochondrial dysfunction.
Parecoxib Inhibited OGD/R-Induced Mitochondrial Dysfunction Bcl-2 is a mitochondira-related protein that protects against neuronal cell death by maintaining the function of mitochondria [17, 18], suggesting that parecoxib’s role in neuroprotection may be related to the maintenance of mitochondria. To investigate whether parecoxib’s neuroprotection against OGD/R was accompanied by the
Discussion The purpose of the current study was to investigate the neuroprotective effects of parecoxib on cerebral ischemia/ reperfusion injury using in vitro OGD/R model. In the
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Fig. 4 Effect of parecoxib on OGD/R inhibited the expression of p-CREB and Bcl-2. a Representative Western blot showed that parecoxib rescued the inhibition of OGD/R on the expression of p-CREB and Bcl-2. b-actin served as an equal loading control. b Quantitative analysis of p-CREB (fold of control). Data are expressed as mean ± SEM (n = 3). **P \ 0.01 compared with Control (PBS), #P \ 0.05 compared with Parecoxib (0 lM) plus OGD4 h/R20 h. c Quantitative analysis of Bcl-2 (fold of control). Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with Control (PBS), #P \ 0.05 compared with Parecoxib (0 lM) plus OGD4 h/R20 h
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Fig. 5 Effect of Bcl-2-shRNA on parecoxib’s neuroprotection against OGD/R-induced neuronal cell death. a Representative Western blot showed that Bcl-2-shRNA inhibits the expression of Bcl-2. b-actin served as an equal loading control. b Neurotoxicity was measured by LDH assay at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment under normaxia condition, #P \ 0.05 compared with PBS plus OGD4 h/ R20 h, &P \ 0.05 compared with parecoxib (20 lM) plus OGD4 h/ R20 h. c MTT reduction was measured by MTT assay at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment under normaxia condition, # P \ 0.05 compared with PBS plus OGD4 h/R20 h, &P \ 0.05 compared with parecoxib (20 lM) plus OGD4 h/R20 h
present study, for the first time, we have demonstrated that parecoxib is neuroprotective against OGD/R-induced neurotoxicity in cultured mouse primary cortical neurons. In
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Fig. 6 Effect of parecoxib on OGD/R induced mitochondria dysfunction. a Mitochondrial membrane potential (DWm) was measured at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment under normaxia condition, #P \ 0.05 compared with PBS plus
OGD4 h/R20 h. b Intracellular ATP levels was measured at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment under normaxia condition, #P \ 0.05 compared with PBS plus OGD4 h/R20 h
addition, we found that parecoxib inhibited OGD/R-induced neurotoxicity via upregulation of CREB phosphorylation and Bcl-2 expression. Furthermore, our results suggested that parecoxib’s neuroprotection was closely related to the improvement of mitochondrial function. It is known that COX-2 is involved in the cerebral ischemia/reperfusion injury, as inhibition of COX-2 results in the reduction of brain injury in response to global ischemia and excitotoxicity [19], and COX-2-deficient mice have ameliorated hippocampal neuronal damage after transient forebrain ischemia [20]. Therefore, it is reasonable that parecoxib, a clinically used inhibitor of COX-2, is capable of protecting against cerebral ischemia/reperfusion injury. Indeed, it has been reported that parecoxib confer neuroprotective effects in transient middle cerebral artery occlusion (tMACO) [6] and permanent middle cerebral artery occlusion (pMACO) rat model [7]. Consistent with these studies, our finding suggested that parecoxib exerts direct neuroprotective effects on primary cortical neurons suffered from OGD/R, as treatment of parecoxib prevented the increase of neuronal cell death over the course of reoxygenation, determined by MTT and LDH assay. Therefore, although parecoxib’s neuroprotective effect is largely relied on the inhibition of inflammation induced by cerebral ischemia/reperfusion in neurovascular, inhibition of OGD/R-induced neurotoxicity is at least, partially responsible for it’s neuroprotection. Cleaved caspase-3 is a key executor in the process of apoptosis of neurons [14], which is induced by OGD/R in the present study. However, treatment of parecoxib significantly inhibited OGD/R-induced expression of cleaved caspase-3. This result indicated that parecoxib’s neuroprotective effect was closely associated with inhibition of neuronal apoptosis, which was consistent with previous
study that caspase-3 immunoreacivity was reduced by parecoxib in the cerebral ischemic penumabra in rats [8]. In addition, Akt/GSK-3b signal had been reported to be involved in parecoxib-induced attenuation of postischemic neuronal apoptosis [8]. However, the effectors of neuronal survival that mediated parecoxib’s neuroprotection against OGD-induced apoptosis still remain to be further investigated. As Akt pathway is responsible for Bcl-2 upregulation via activation of the CREB transcription factor [21], we thus sought to investigate whether CREB/Bcl-2 axis mediated neuroprotective effect of parecoxib. Consistent with this, CREB phosphorylation at Ser133 and Bcl-2 expression by parecoxib can be induced in a dose dependent manner, and OGD/R inhibited CREB phosphorylation on Ser133 and Bcl-2 expression can be partially recovered by parecoxib. These results support the important role of CREB phosphorylation and Bcl-2 expression in parecoxib’s neuroprotection. However, we can not conclude that the neuroprotective role of parecoxib against OGD/R is solely relied on the CREB/Bcl-2 axis. Previous studies suggested that neuroglobin, a key target of CREB [22], can also protect against OGD/R-induced neurotoxicity by amelioration of mitochondrial function [12]. Therefore, Bcl-2 might only partially contribute to parecoxib-mediated neuroprotection. Recent studies suggested that endoplasmic reticulum (ER) stress related proteins including GRP78, ORP150, CHOP and Foxo1 were involved in parecoxib-mediated neuroprotection, suggesting that inhibition of ER stress responses may contribute to parecoxib-mediated neuroprotection [23]. In our study, we further identified that parecoxib could protect against OGD/R by upregulating Bcl-2, an important mitochondria-related protein. In addition, our results showed that parecoxib can rescue OGD/R
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induced mitochondrial dysfunction. Therefore, parecoxib protected against OGD/R induced neurotoxicity, is probably associated with the inhibition of both ER and mitochondria-mediated neuronal cell death. Interestingly, accumulating evidence suggested that ER was involved in regulating apoptosis, either independent of mitochondrial, or in concert with mitochondria-initiated pathways [24]. Moreover, it is worth noting that Bcl-2 protein can localize and target to the mitochondria and ER both, and promote neuron survival [25]. It is possible that Bcl-2, as a key effector of parecoxib, inhibits OGD/R induced neuronal apoptosis via cross-talk between ER and mitochondriadependent death cascades. This issue will be further elucidated in our future study. In conclusion, our study demonstrated the neuroprotective role of parecoxib against OGD/R-induced neuronal injury. Our results also indicated that these neuroprotective effects were at least partially mediated by the increase of Bcl-2, and the maintenance of mitochondrial function. We confidently believe that the present study will not only aid understanding the role and mechanisms of parecoxib in protecting against cerebral ischemia–reperfusion injury, but also help in the development of therapeutic strategies to reduce neuronal cell damage after ischemia. Acknowledgments This work was supported by the National Natural Science of China (No. 81201018). Conflict of interest
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We declare that we have no conflict of interest. 17.
References 1. Majid A (2014) Neuroprotection in stroke: past, present, and future. ISRN Neurol 2014:515716 2. Reed SD, Cramer SC, Blough DK, Meyer K, Jarvik JG (2001) Treatment with tissue plasminogen activator and inpatient mortality rates for patients with ischemic stroke treated in community hospitals. Stroke 32:1832–1840 3. Kleinig TJ, Vink R (2009) Suppression of inflammation in ischemic and hemorrhagic stroke: therapeutic options. Curr Opin Neurol 22:294–301 4. Hawkey CJ, Fortun PJ (2005) Cyclooxygenase-2 inhibitors. Curr Opin Gastroenterol 21:660–664 5. Wei W, Zhao T, Li Y (2013) Efficacy and safety of parecoxib sodium for acute postoperative pain: a meta-analysis. Exp Ther Med 6:525–531 6. Kelsen J, Kjaer K, Chen G, Pedersen M, Rohl L, Frokiaer J, Nielsen S, Nyengaard JR, Ronn LC (2006) Parecoxib is neuroprotective in spontaneously hypertensive rats after transient middle cerebral artery occlusion: a divided treatment response? J Neuroinflamm 3:31 7. Wang N, Guo QL, Ye Z, Xia PP, Wang E, Yuan YJ (2011) Preconditioning of intravenous parecoxib attenuates focal cerebral ischemia/reperfusion injury in rats. Chin Med J (Engl) 124:2004–2008 8. Ye Z, Wang N, Xia P, Wang E, Yuan Y, Guo Q (2012) Delayed administration of parecoxib, a specific COX-2 inhibitor,
123
18.
19.
20.
21.
22.
23.
attenuated postischemic neuronal apoptosis by phosphorylation Akt and GSK-3beta. Neurochem Res 37:321–329 Cimarosti H, Henley JM (2008) Investigating the mechanisms underlying neuronal death in ischemia using in vitro oxygenglucose deprivation: potential involvement of protein SUMOylation. Neuroscientist 14:626–636 Liu D, Lu C, Wan R, Auyeung WW, Mattson MP (2002) Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release. J Cereb Blood Flow Metab 22:431–443 Liu J, Yu Z, Guo S, Lee SR, Xing C, Zhang C, Gao Y, Nicholls DG, Lo EH, Wang X (2009) Effects of neuroglobin overexpression on mitochondrial function and oxidative stress following hypoxia/reoxygenation in cultured neurons. J Neurosci Res 87:164–170 Yu Z, Liu N, Li Y, Xu J, Wang X (2013) Neuroglobin overexpression inhibits oxygen-glucose deprivation-induced mitochondrial permeability transition pore opening in primary cultured mouse cortical neurons. Neurobiol Dis 56:95–103 Velier JJ, Ellison JA, Kikly KK, Spera PA, Barone FC, Feuerstein GZ (1999) Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat. J Neurosci 19:5932–5941 Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA et al (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37–43 Carloni S, Girelli S, Scopa C, Buonocore G, Longini M, Balduini W (2010) Activation of autophagy and Akt/CREB signaling play an equivalent role in the neuroprotective effect of rapamycin in neonatal hypoxia-ischemia. Autophagy 6:366–377 Lin CH, Chen PS, Gean PW (2008) Glutamate preconditioning prevents neuronal death induced by combined oxygen-glucose deprivation in cultured cortical neurons. Eur J Pharmacol 589:85–93 Fuenzalida K, Quintanilla R, Ramos P, Piderit D, Fuentealba RA, Martinez G, Inestrosa NC, Bronfman M (2007) Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis. J Biol Chem 282:37006–37015 Soane L, Fiskum G (2005) Inhibition of mitochondrial neural cell death pathways by protein transduction of Bcl-2 family proteins. J Bioenerg Biomembr 37:179–190 Nagayama M, Niwa K, Nagayama T, Ross ME, Iadecola C (1999) The cyclooxygenase-2 inhibitor NS-398 ameliorates ischemic brain injury in wild-type mice but not in mice with deletion of the inducible nitric oxide synthase gene. J Cereb Blood Flow Metab 19:1213–1219 Sasaki T, Kitagawa K, Yamagata K, Takemiya T, Tanaka S, Omura-Matsuoka E, Sugiura S, Matsumoto M, Hori M (2004) Amelioration of hippocampal neuronal damage after transient forebrain ischemia in cyclooxygenase-2-deficient mice. J Cereb Blood Flow Metab 24:107–113 Perianayagam MC, Madias NE, Pereira BJ, Jaber BL (2006) CREB transcription factor modulates Bcl-2 transcription in response to C5a in HL-60-derived neutrophils. Eur J Clin Invest 36:353–361 Liu N, Yu Z, Li Y, Yuan J, Zhang J, Xiang S, Wang X (2013) Transcriptional regulation of mouse neuroglobin gene by cyclic AMP responsive element binding protein (CREB) in N2a cells. Neurosci Lett 534:333–337 Ye Z, Wang N, Xia P, Wang E, Liao J, Guo Q (2013) Parecoxib suppresses CHOP and Foxo1 nuclear translocation, but increases
Neurochem Res GRP78 levels in a rat model of focal ischemia. Neurochem Res 38:686–693 24. Ghribi O, DeWitt DA, Forbes MS, Herman MM, Savory J (2001) Co-involvement of mitochondria and endoplasmic reticulum in regulation of apoptosis: changes in cytochrome c, Bcl-2 and Bax in the hippocampus of aluminum-treated rabbits. Brain Res 903:66–73
25. Renton JP, Xu N, Clark JJ, Hansen MR (2010) Interaction of neurotrophin signaling with Bcl-2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth. J Neurosci Res 88:2239–2251
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ORIGINAL PAPER
Parecoxib Protects Mouse Cortical Neurons Against OGD/R Induced Neurotoxicity by Up-Regulating Bcl-2 Yueling Wang1 • Wenjuan Ma1 • Aijun Jia2 • Qulian Guo1
Received: 2 January 2015 / Revised: 6 April 2015 / Accepted: 9 April 2015 Ó Springer Science+Business Media New York 2015
Abstract Ischemic stroke remains a significant problem that is the major cause of death and disability worldwide. Parecoxib is clinically used for short-term management of postoperative pain. Administration of parecoxib in rats has been reported to protect against the cerebral ischemia/ reperfusion. However, the neuroprotective mechanism of parecoxib is still largely unknown. In this study, we found parecoxib could protect against neurotoxicity induced by 4 h oxygen–glucose deprivation (OGD) plus reoxgenation for 20 h, a widely used in vitro model of ischemia/reperfusion. In addition, we characterized the molecular mechanism of parecoxib’s neuroprotection. We found parecoxib was able to activate CREB, and subsequently maintained the expression of Bcl-2, which is an important mitochondria-associated protein. Inhibition of endogenous Bcl-2 expression by transfection of Bcl-2-shRNA significantly attenuated the neuroprotective effects of parecoxib treatment. Furthermore, ATP production assay and mitochondrial membrane potential (DWm) assay suggested that parecoxib exerted neuroprotective effect against OGD/ R by maintaining the function of mitochondria. These data suggested that parecoxib treatment is a potential therapeutic approach for protecting against ischemia/reperfusion injury.
& Qulian Guo [email protected] 1
Department of Anesthesiology, Xiangya Hospital of Central South University, 87 Xiangya Road, Changsha City 410008, Hunan Province, China
2
Department of Respiratory Medicine, The Second Xiangya Hospital, Central South University, Changsha City, Hunan Province, China
Keywords Parecoxib Oxygen–glucose deprivation/ reoxgenation Neurotoxicity Bcl-2 Abbreviations OGD/R Oxygen–glucose deprivation/reoxgenation CREB cAMP-response element binding protein COX-2 Cyclooxygenase-2 LDH Lactate dehydrogenase MACO Middle cerebral artery occlusion
Introduction Ischemic stroke is the common cause of mortality and disability worldwide. Although great efforts have been made in the past decades, the therapeutic methods for patients with ischemic stroke remain limited [1]. Until now, only one pharmacological agent, named recombinant tissue plasminogen activator (rtPA), has been approved by US Food and Drug Administration (FDA) for acute ischemic stroke by breaking down the occluding clot in cerebral vessels and restoring blood flow to the ischemic brain [2]. However, cerebral ischemia/reperfusion can often result in inflammation by upregulation of proinflammatory cytokines including interleukin-1b (IL-1b) and tumor necrosis factora (TNF-a) and cause secondary damage [3]. Therefore, the anti-inflammatory agents are potentially useful for protecting against the cerebral ischemia/reperfusion. Cyclooxygenase-2 (COX-2) is an enzyme that mediates inflammatory process through protaglandins release and inhibition of COX-2 thereby relieves inflammation and pain [4]. Parecoxib, a novel potent and selective COX-2 inhibitor, has been widely used for short-term management of postoperative pain in clinical parctice. Clinical trials
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have indicated that parecoxib is effective and well tolerated for treatment of postoperative pain after dental surgery, orthopedic surgery [5]. Due to the anti-inflammatory property, the neuroprotective effect of parecoxib had been studied previously. An earlier study reported that parecoxib is neuroprotective in spontaneously hypertensive rats after transient middle cerebral artery occlusion, which is closely related to the inflammation inhibition [6]. Another study found that pre-treatment with intravenous parecoxib protects against focal cerebral ischemia/reperfusion injury, and the neuroprotective effects is probably associated with the inhibition of inflammatory reaction [7]. Moreover, our previous study suggested that parecoxib plays neuroprotective role by attenuating postischemic neuronal apoptosis, which mediated by activation of Akt and GSK-3b signaling pathway [8]. However, the underlying neuroprotective mechanisms of parecoxib against cerebral ischemia/reperfusion injury still remain largely unknown. Oxygen–glucose deprivation (OGD) is a classical in vitro model of ischemia, which has been frequently used for mimicking the interruption of the blood supply including oxygen and nutrients to the brain using primary neuronal cultures [9]. In this study, we aimed to investigate the role of parecoxib in the protection against cerebral ischemia/reperfusion injury using OGD plus reoxygenation (OGD/R) model. We found that parecoxib treatment was capable of protecting against OGD/Rinduced neuronal injury in mouse primary cortical neurons. Moreover, we found parecoxib could induce the activation of CREB and the expression of Bcl-2, which is responsible for the neuroprotective effect of parecoxib. Importantly, Bcl-2 protein had been demonstrated to exert neuroprotective effect by maintaining the function of mitochondria [10], we thus examined the role of Bcl-2 in parecoxib’s neuroprotection against OGD/R-induced mitochondria injury. Our results suggested that the neuroprotective effect of parecoxib was closely associated with the functional recovery of mitochondria. The results of present study suggested that parecoxib treatment is potentially a therapeutic approach for intervention of ischemia/ reperfusion injury.
Materials and Methods All animal procedures were carried out following protocols approved by the Ethic Committee of the Xiangya Hosipital of Central South University.
Materials Dulbecco’s modification of Eagle’s medium (DMEM), Neurobasal medium (NBM), glutamine, B27 supplements, LipofectamineTM 2000 were purchased from Invitrogen.
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Fetal bovine serum and 0.05 % trypsin–EDTA were purchased from Gibco. Antibodies against Akt, phosphoSer473-Akt (p-Akt), CREB, phospho-Ser133-CREB (pCREB), caspase 3, cleaved caspase 3 and Bcl-2 were purchased from Cell Signaling Technology. Antibodies against NeuN and GFAP were purchased from Abcam. bactin antibody was obtained from Sigma-Aldrich. Primary Mouse Cortical Neuron Culture Primary mouse cortical neuron cultures were established from C57 BL/6 mouse embryos using methods described before [11]. Briefly, primary mouse cortical neurons were isolated from 15 day mouse embryonic cortex. Dissociated neurons were seeded into 6 or 24-well plates, and maintained in NBM containing 0.3 mM glutamine and 3 % B27 supplements. The NBM was half changed with fresh NBM each 3–4 days. Bcl-2 Knockdown Using Bcl-2-shRNA Bcl-2-shRNA and scrambled shRNA (control shRNA) were inserted in pGFP-V-RS retroviral vector, respectively. The vectors were then transfected into primary cortical neurons according to the manufacturer’s instruction (OriGene). Briefly, for 3 9 105 cells, 1 lg of Bcl-2-shRNA or control shRNA vector was pre-mixed with 3 lL of TurboFectin solution for 30 min, and the mixture was added into NBM of neurons for incubation in a 5 % CO2 incubator for another 48 h. Oxygen–Glucose Deprivation (OGD) Plus Reoxygenation The NBM of days 8 of primary neuron cultures was replaced by serum free DMEM, and the cells were then put in a modular chamber (Billups–Rothenber) perfused with 90 % N2/5 % CO2/5 % H2 for 4 h at 37°C. Following OGD, cells were transferred into the incubator for reoxygenation for 4 h, then treated with parecoxib and followed by reoxygenation for another 16 h (The total reoxygenation time is 20 h). The control group were underwent media change, but were not exposed to OGD and reoxygenation. Lactate Dehydrogenase (LDH) Assay After 4 h OGD plus 20 h reoxygenation, LDH assay was performed to measure neurotoxicity using the LDH detection kit according to the manufacturer’s instruction (Roche). The LDH activity in the untreated normal cell culture medium was detected as ‘‘low control’’, and the LDH activity released from the untreated control neurons was determined as ‘‘high control’’ when the neurons were
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treated with 0.5 % Triton X-100. To determine the percentage of neurotoxicity, the resulting values are substituted in the following equation: Neurotoxicity (%) = (experimental value - low control)/(high control - low control) 9 100.
with 5 lM JC-1 for 20 min at 37°C in the incubator. The flurescent signals of DWm with excitation 545 nm and emission 620 nm were read with the microplate reader (Lifecare Medical). Statistical Analysis
MTT Assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to detect cell viability. Briefly, cells were treated with MTT which was dissolved in DMEM at a final concentration of 1 mg/ml, and incubated at 37°C for 4 h. Supernatant was removed and replaced with 100 ll of DMSO. The optical density (OD) was recorded on the microplate reader (Lifecare Medical) at 570 nm. Cell viability was expressed as a percentage of the control value.
All data were expressed as mean ± SEM. Statistical significance was determined by a one-way ANOVA followed by Tukey–Kramer tests. P value \0.05 was considered significant. At least three independent experiments were performed.
Results Parecoxib Protects Against OGD/R-Induced Neuronal Cell Death
Western Blot Cells were lysed using cell lysis buffer (Cell Signaling Technology). Equal amounts of proteins from neurons were subjected to 4–12 % SDS-PAGE gel and transferred to PVDF membranes. The blot was blocked with 5 % nonfat dried milk, incubated overnight at 4°C with mouse primary antibody. Then, incubation was followed by horseradish peroxidase-conjugated anti-mouse secondary antibodies. Immunolabeling was performed using enhanced chemiluminescence (Amersham Pharmacia Biotech) following manufacturer’s instruction, and then exposed to film (Eastman Kodak Co.). ATP Production Assay ATP production assay was performed to determine the intracellular ATP levels in mouse primary cortical neurons using ATP Bioluminescence assay kit according to manufacturer’s instructions (Roche). After treatment, neurons were lysed with 50 lL cell lysis buffer and shaked for 2 min to ensure completely release of ATP. 5 lL cell lysates were used for protein concentration measurement, the rest of the cell lysates were added with 100 lL luciferase reagent. Then the luminescence was measured by VeritasTM Microplate Luminometer (Turner BioSystems).
To investigate the effect of parecoxib on OGD/R-induced neuronal cell death in cultured primary cortical neurons, the MTT assay was used. Consistent with previous study [12], OGD4 h/R20 h induced a striking increase in cell death, whereas treatment with parecoxib at 4 h of reoxgenation after OGD 4 h resulted in a significantly improvement in the survival rate of neurons, in a dosedependent manner (Fig. 1a). Similarly, in LDH assay, OGD/R largely increased LDH release, but treatment with parecoxib significantly decreased LDH release, thereby indicating that parecoxib can attenuate OGD/R-induced neuronal damage (Fig. 1b). Parecoxib Attenuated OGD/R-Induced Cleavage of Caspase-3 Caspase-3 is a pro-apoptotic protein that is usually used as the maker for cell apoptosis [13, 14]. To investigate the effect of parecoxib on the OGD/R-induced activation of caspase-3, western blot was performed. The results showed that OGD/R significantly increased the cleavage of caspase-3, whereas treatment with parecoxib significantly decreased OGD/R-induced the expression of cleaved caspase-3 when compared with control group, indicating that parecoxib was capable of inhibiting OGD/R-induced neuronal apoptosis (Fig. 2).
Mitochondrial Membrane Potential Mitochondrial membrane potential (DWm) was measured using JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide) mitochondrial membrane potential assay kit according to the manufacturer’s instructions (Cell Technology). After 4 h OGD plus reoxygenation for 20 h, the culture neurons were incubated
Parecoxib Significantly Increases CREB Phosphorylation and Bcl-2 Expression Our previous study indicated that parecoxib was able to increase the expression of Akt phosphorylation (p-Akt) [8], and Akt/CREB signaling played an important role in protecting against ischemia/reperfusion injury [15], the effect
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Fig. 1 Effect of dose-dependent of parecoxib on OGD induced neurotoxicity in mouse primary cortical neurons. a Cell viability was measured by MTT assay at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment alone, #P \ 0.05 compared with PBS plus OGD4 h/
R20 h. b Neurotoxicity was measured by LDH assay at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment alone, #P \ 0.05 compared with PBS plus OGD4 h/R20 h
Fig. 2 Effect of parecoxib on OGD/R induced the cleavage of Caspase-3. Representative Western blot showed that parecoxib inhibited OGD/R induced expression of cleaved Caspase-3. b-actin served as an equal loading control. Quantitative analysis of cleaved
Caspase-3 (fold of control). Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with Control (PBS), #P \ 0.05 compared with parecoxib (0 lM) plus OGD4 h/R20 h
of parecoxib on the p-Akt, p-CREB and a CREB target Bcl-2 protein were detected. As expected, the results showed that parecoxib significantly increased the expression levels of p-Akt, p-CREB and Bcl-2 in a dose-dependent manner (Fig. 3).
shown, OGD/R resulted in the downregulation of CREB phosphorylation and Bcl-2 expression, whereas treatment of parecoxib significantly rescued the inhibitory effect of OGD/R on CREB phosphorylation and Bcl-2 expression (Fig. 4). Therefore, these data implied that Bcl-2 is of significant importance for the neuroprotection role of parecoxib against OGD/R.
Parecoxib Partially Rescued the Inhibitory Effect of OGD/R on CREB Phosphorylation and Bcl-2 Expression OGD/R had been demonstrated to inhibit CREB phosphorylation and Bcl-2 expression [16]. To investigate the role of parecoxib in OGD/R mediated inhibition of CREB phosphorylation and Bcl-2 expression, mouse primary cortical neurons were suffered from OGD for 4 h, and incubated with or without parecoxib at 4 h of reoxygenation, followed by reoxgenation for another 16 h. As previously
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Parecoxib Inhibited OGD/R-Induced Neuronal Injury by Upregulating Bcl-2 Expression To further investigate the effect of Bcl-2 knockdown on the neuroprotective role of parecoxib, Bcl-2-shRNA was transfected into primary cortical neurons. Western blot showed that transfection of Bcl-2-shRNA, but not controlshRNA, led to markedly reduction of Bcl-2 protein expression in primary cortical neurons (Fig. 5a). In LDH
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Fig. 3 Effect of dose-dependent of parecoxib on the expression of p-Akt, p-CREB and Bcl-2. a Representative Western blot showed that dose-dependent of parecoxib induced the expression of p-Akt, p-CREB and Bcl-2. b-Actin served as an equal loading control. b Quantitative analysis of p-Akt (fold of control). Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with Parecoxib
(0 lM). c Quantitative analysis of p-CREB (fold of control). Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with Parecoxib (0 lM). d Quantitative analysis of Bcl-2 (fold of control). Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with Parecoxib (0 lM)
assay, we found that inhibition of endogenous Bcl-2 expression by specific Bcl-2 shRNA in primary cortical neurons significantly attenuated the role of parecoxib against OGD/R-induced neurotoxicity (Fig. 5b). Similar results were also found in MTT assay, transfection with Bcl-2-shRNA significantly inhibited the neuroprotective effect of parecoxib (Fig. 5c). Therefore, these results suggested that parecoxib inhibited OGD/R-induced neuronal injury at least in part through a mechanism involving increased Bcl-2 expression.
improvement of mitochondrial function, mitochondrial membrane potential (DWm) was measured using JC-1 reagents. As expected, DWm value was largely decreased by OGD/R compared to normal neurons, while the reduction was significantly rescued by parecoxib (Fig. 6a). In addition, intracellular ATP levels in neurons were also detected, as it’s another functional marker of mitochondria. Our results showed that intracellular ATP levels in neurons were significantly decreased by OGD/R, but parecoxib treatment led to significant improvement of ATP levels (Fig. 6b). Taken together, these data suggested that parecoxib could inhibit OGD/R-induced mitochondrial dysfunction.
Parecoxib Inhibited OGD/R-Induced Mitochondrial Dysfunction Bcl-2 is a mitochondira-related protein that protects against neuronal cell death by maintaining the function of mitochondria [17, 18], suggesting that parecoxib’s role in neuroprotection may be related to the maintenance of mitochondria. To investigate whether parecoxib’s neuroprotection against OGD/R was accompanied by the
Discussion The purpose of the current study was to investigate the neuroprotective effects of parecoxib on cerebral ischemia/ reperfusion injury using in vitro OGD/R model. In the
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Fig. 4 Effect of parecoxib on OGD/R inhibited the expression of p-CREB and Bcl-2. a Representative Western blot showed that parecoxib rescued the inhibition of OGD/R on the expression of p-CREB and Bcl-2. b-actin served as an equal loading control. b Quantitative analysis of p-CREB (fold of control). Data are expressed as mean ± SEM (n = 3). **P \ 0.01 compared with Control (PBS), #P \ 0.05 compared with Parecoxib (0 lM) plus OGD4 h/R20 h. c Quantitative analysis of Bcl-2 (fold of control). Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with Control (PBS), #P \ 0.05 compared with Parecoxib (0 lM) plus OGD4 h/R20 h
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Fig. 5 Effect of Bcl-2-shRNA on parecoxib’s neuroprotection against OGD/R-induced neuronal cell death. a Representative Western blot showed that Bcl-2-shRNA inhibits the expression of Bcl-2. b-actin served as an equal loading control. b Neurotoxicity was measured by LDH assay at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment under normaxia condition, #P \ 0.05 compared with PBS plus OGD4 h/ R20 h, &P \ 0.05 compared with parecoxib (20 lM) plus OGD4 h/ R20 h. c MTT reduction was measured by MTT assay at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment under normaxia condition, # P \ 0.05 compared with PBS plus OGD4 h/R20 h, &P \ 0.05 compared with parecoxib (20 lM) plus OGD4 h/R20 h
present study, for the first time, we have demonstrated that parecoxib is neuroprotective against OGD/R-induced neurotoxicity in cultured mouse primary cortical neurons. In
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Fig. 6 Effect of parecoxib on OGD/R induced mitochondria dysfunction. a Mitochondrial membrane potential (DWm) was measured at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment under normaxia condition, #P \ 0.05 compared with PBS plus
OGD4 h/R20 h. b Intracellular ATP levels was measured at 4 h OGD plus 20 h reoxygenation. Data are expressed as mean ± SEM (n = 3). *P \ 0.05 compared with PBS treatment under normaxia condition, #P \ 0.05 compared with PBS plus OGD4 h/R20 h
addition, we found that parecoxib inhibited OGD/R-induced neurotoxicity via upregulation of CREB phosphorylation and Bcl-2 expression. Furthermore, our results suggested that parecoxib’s neuroprotection was closely related to the improvement of mitochondrial function. It is known that COX-2 is involved in the cerebral ischemia/reperfusion injury, as inhibition of COX-2 results in the reduction of brain injury in response to global ischemia and excitotoxicity [19], and COX-2-deficient mice have ameliorated hippocampal neuronal damage after transient forebrain ischemia [20]. Therefore, it is reasonable that parecoxib, a clinically used inhibitor of COX-2, is capable of protecting against cerebral ischemia/reperfusion injury. Indeed, it has been reported that parecoxib confer neuroprotective effects in transient middle cerebral artery occlusion (tMACO) [6] and permanent middle cerebral artery occlusion (pMACO) rat model [7]. Consistent with these studies, our finding suggested that parecoxib exerts direct neuroprotective effects on primary cortical neurons suffered from OGD/R, as treatment of parecoxib prevented the increase of neuronal cell death over the course of reoxygenation, determined by MTT and LDH assay. Therefore, although parecoxib’s neuroprotective effect is largely relied on the inhibition of inflammation induced by cerebral ischemia/reperfusion in neurovascular, inhibition of OGD/R-induced neurotoxicity is at least, partially responsible for it’s neuroprotection. Cleaved caspase-3 is a key executor in the process of apoptosis of neurons [14], which is induced by OGD/R in the present study. However, treatment of parecoxib significantly inhibited OGD/R-induced expression of cleaved caspase-3. This result indicated that parecoxib’s neuroprotective effect was closely associated with inhibition of neuronal apoptosis, which was consistent with previous
study that caspase-3 immunoreacivity was reduced by parecoxib in the cerebral ischemic penumabra in rats [8]. In addition, Akt/GSK-3b signal had been reported to be involved in parecoxib-induced attenuation of postischemic neuronal apoptosis [8]. However, the effectors of neuronal survival that mediated parecoxib’s neuroprotection against OGD-induced apoptosis still remain to be further investigated. As Akt pathway is responsible for Bcl-2 upregulation via activation of the CREB transcription factor [21], we thus sought to investigate whether CREB/Bcl-2 axis mediated neuroprotective effect of parecoxib. Consistent with this, CREB phosphorylation at Ser133 and Bcl-2 expression by parecoxib can be induced in a dose dependent manner, and OGD/R inhibited CREB phosphorylation on Ser133 and Bcl-2 expression can be partially recovered by parecoxib. These results support the important role of CREB phosphorylation and Bcl-2 expression in parecoxib’s neuroprotection. However, we can not conclude that the neuroprotective role of parecoxib against OGD/R is solely relied on the CREB/Bcl-2 axis. Previous studies suggested that neuroglobin, a key target of CREB [22], can also protect against OGD/R-induced neurotoxicity by amelioration of mitochondrial function [12]. Therefore, Bcl-2 might only partially contribute to parecoxib-mediated neuroprotection. Recent studies suggested that endoplasmic reticulum (ER) stress related proteins including GRP78, ORP150, CHOP and Foxo1 were involved in parecoxib-mediated neuroprotection, suggesting that inhibition of ER stress responses may contribute to parecoxib-mediated neuroprotection [23]. In our study, we further identified that parecoxib could protect against OGD/R by upregulating Bcl-2, an important mitochondria-related protein. In addition, our results showed that parecoxib can rescue OGD/R
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induced mitochondrial dysfunction. Therefore, parecoxib protected against OGD/R induced neurotoxicity, is probably associated with the inhibition of both ER and mitochondria-mediated neuronal cell death. Interestingly, accumulating evidence suggested that ER was involved in regulating apoptosis, either independent of mitochondrial, or in concert with mitochondria-initiated pathways [24]. Moreover, it is worth noting that Bcl-2 protein can localize and target to the mitochondria and ER both, and promote neuron survival [25]. It is possible that Bcl-2, as a key effector of parecoxib, inhibits OGD/R induced neuronal apoptosis via cross-talk between ER and mitochondriadependent death cascades. This issue will be further elucidated in our future study. In conclusion, our study demonstrated the neuroprotective role of parecoxib against OGD/R-induced neuronal injury. Our results also indicated that these neuroprotective effects were at least partially mediated by the increase of Bcl-2, and the maintenance of mitochondrial function. We confidently believe that the present study will not only aid understanding the role and mechanisms of parecoxib in protecting against cerebral ischemia–reperfusion injury, but also help in the development of therapeutic strategies to reduce neuronal cell damage after ischemia. Acknowledgments This work was supported by the National Natural Science of China (No. 81201018). Conflict of interest
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We declare that we have no conflict of interest. 17.
References 1. Majid A (2014) Neuroprotection in stroke: past, present, and future. ISRN Neurol 2014:515716 2. Reed SD, Cramer SC, Blough DK, Meyer K, Jarvik JG (2001) Treatment with tissue plasminogen activator and inpatient mortality rates for patients with ischemic stroke treated in community hospitals. Stroke 32:1832–1840 3. Kleinig TJ, Vink R (2009) Suppression of inflammation in ischemic and hemorrhagic stroke: therapeutic options. Curr Opin Neurol 22:294–301 4. Hawkey CJ, Fortun PJ (2005) Cyclooxygenase-2 inhibitors. Curr Opin Gastroenterol 21:660–664 5. Wei W, Zhao T, Li Y (2013) Efficacy and safety of parecoxib sodium for acute postoperative pain: a meta-analysis. Exp Ther Med 6:525–531 6. Kelsen J, Kjaer K, Chen G, Pedersen M, Rohl L, Frokiaer J, Nielsen S, Nyengaard JR, Ronn LC (2006) Parecoxib is neuroprotective in spontaneously hypertensive rats after transient middle cerebral artery occlusion: a divided treatment response? J Neuroinflamm 3:31 7. Wang N, Guo QL, Ye Z, Xia PP, Wang E, Yuan YJ (2011) Preconditioning of intravenous parecoxib attenuates focal cerebral ischemia/reperfusion injury in rats. Chin Med J (Engl) 124:2004–2008 8. Ye Z, Wang N, Xia P, Wang E, Yuan Y, Guo Q (2012) Delayed administration of parecoxib, a specific COX-2 inhibitor,
123
18.
19.
20.
21.
22.
23.
attenuated postischemic neuronal apoptosis by phosphorylation Akt and GSK-3beta. Neurochem Res 37:321–329 Cimarosti H, Henley JM (2008) Investigating the mechanisms underlying neuronal death in ischemia using in vitro oxygenglucose deprivation: potential involvement of protein SUMOylation. Neuroscientist 14:626–636 Liu D, Lu C, Wan R, Auyeung WW, Mattson MP (2002) Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release. J Cereb Blood Flow Metab 22:431–443 Liu J, Yu Z, Guo S, Lee SR, Xing C, Zhang C, Gao Y, Nicholls DG, Lo EH, Wang X (2009) Effects of neuroglobin overexpression on mitochondrial function and oxidative stress following hypoxia/reoxygenation in cultured neurons. J Neurosci Res 87:164–170 Yu Z, Liu N, Li Y, Xu J, Wang X (2013) Neuroglobin overexpression inhibits oxygen-glucose deprivation-induced mitochondrial permeability transition pore opening in primary cultured mouse cortical neurons. Neurobiol Dis 56:95–103 Velier JJ, Ellison JA, Kikly KK, Spera PA, Barone FC, Feuerstein GZ (1999) Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat. J Neurosci 19:5932–5941 Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA et al (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37–43 Carloni S, Girelli S, Scopa C, Buonocore G, Longini M, Balduini W (2010) Activation of autophagy and Akt/CREB signaling play an equivalent role in the neuroprotective effect of rapamycin in neonatal hypoxia-ischemia. Autophagy 6:366–377 Lin CH, Chen PS, Gean PW (2008) Glutamate preconditioning prevents neuronal death induced by combined oxygen-glucose deprivation in cultured cortical neurons. Eur J Pharmacol 589:85–93 Fuenzalida K, Quintanilla R, Ramos P, Piderit D, Fuentealba RA, Martinez G, Inestrosa NC, Bronfman M (2007) Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis. J Biol Chem 282:37006–37015 Soane L, Fiskum G (2005) Inhibition of mitochondrial neural cell death pathways by protein transduction of Bcl-2 family proteins. J Bioenerg Biomembr 37:179–190 Nagayama M, Niwa K, Nagayama T, Ross ME, Iadecola C (1999) The cyclooxygenase-2 inhibitor NS-398 ameliorates ischemic brain injury in wild-type mice but not in mice with deletion of the inducible nitric oxide synthase gene. J Cereb Blood Flow Metab 19:1213–1219 Sasaki T, Kitagawa K, Yamagata K, Takemiya T, Tanaka S, Omura-Matsuoka E, Sugiura S, Matsumoto M, Hori M (2004) Amelioration of hippocampal neuronal damage after transient forebrain ischemia in cyclooxygenase-2-deficient mice. J Cereb Blood Flow Metab 24:107–113 Perianayagam MC, Madias NE, Pereira BJ, Jaber BL (2006) CREB transcription factor modulates Bcl-2 transcription in response to C5a in HL-60-derived neutrophils. Eur J Clin Invest 36:353–361 Liu N, Yu Z, Li Y, Yuan J, Zhang J, Xiang S, Wang X (2013) Transcriptional regulation of mouse neuroglobin gene by cyclic AMP responsive element binding protein (CREB) in N2a cells. Neurosci Lett 534:333–337 Ye Z, Wang N, Xia P, Wang E, Liao J, Guo Q (2013) Parecoxib suppresses CHOP and Foxo1 nuclear translocation, but increases
Neurochem Res GRP78 levels in a rat model of focal ischemia. Neurochem Res 38:686–693 24. Ghribi O, DeWitt DA, Forbes MS, Herman MM, Savory J (2001) Co-involvement of mitochondria and endoplasmic reticulum in regulation of apoptosis: changes in cytochrome c, Bcl-2 and Bax in the hippocampus of aluminum-treated rabbits. Brain Res 903:66–73
25. Renton JP, Xu N, Clark JJ, Hansen MR (2010) Interaction of neurotrophin signaling with Bcl-2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth. J Neurosci Res 88:2239–2251
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