Inflamm. Res. DOI 10.1007/s00011-014-0710-z
Inflammation Research
ORIGINAL RESEARCH PAPER
Anti-inflammatory effects of curcumin in experimental spinal cord injury in rats Wei Jin • Jing Wang • Tiansheng Zhu • Baoyu Yuan • Hongbin Ni Jian Jiang • Handong Wang • Weibang Liang
•
Received: 4 October 2013 / Revised: 17 December 2013 / Accepted: 12 January 2014 Ó Springer Basel 2014
Abstract Aim Antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) has been shown in our previous studies to play an important role in protection against spinal cord injury (SCI) induced inflammatory response. The objective of this study was to test whether curcumin, a novel Nrf2 activator, can protect the spinal cord against SCI-induced inflammatory damage. Methods Adult male Sprague–Dawley rats were subjected to laminectomy at T8–T9 and compression with a vascular clip. The spinal cords spanning the injury site about 0.8 cm were collected for testing. There were three groups: (a) sham group; (b) SCI group; and (c) SCI ? curcumin group. We measured Nrf2 and nuclear factor kappa B (NF-jB) binding activities by electrophoretic mobility shift assay, concentrations of tumor necrosis factor-a, interleukin-1b and interleukin-6 by enzymelinked immunosorbent assay, hindlimb locomotion function by Basso, Beattie, and Bresnahan rating, spinal cord edema by the wet/dry weight method, and apoptosis by
Responsible Editor: Ji Zhang. W. Jin and J. Wang contributed equally to this work. W. Jin J. Wang T. Zhu B. Yuan H. Ni J. Jiang W. Liang (&) Department of Neurosurgery, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, People’s Republic of China e-mail: [email protected] H. Wang (&) Department of Neurosurgery, Jinling Hospital, Medical School of Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu, People’s Republic of China e-mail: [email protected]
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis. Results Induction of the Nrf2 activity by curcumin markedly decreased NF-jB activation and inflammatory cytokines production in the injured spinal cord. Administration of curcumin also significantly ameliorated the secondary spinal cord damage, as shown by decreased severity of locomotion deficit, spinal cord edema, and apoptosis. Conclusion Post-SCI curcumin administration attenuates the inflammatory response in the injured spinal cord, and this may be a mechanism whereby curcumin improves the outcome following SCI. Keywords Curcumin Spinal cord injury Nrf2 Inflammatory response
Introduction Spinal cord injury (SCI) initiates a series of cellular and molecular cascade events, and a combination of secondary injury factors leads to a progressive neural injury [1]. Among these secondary injury factors, inflammatory response is the major one and plays an important role in regulating the pathogenesis of SCI [2, 3]. Increased levels of inflammation-related factors with the injured spinal cord, including tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), interleukin-6 (IL-6), and their mediator NF-jB, are believed to contribute to the neural damage [4, 5]. Previous studies of our laboratory demonstrated that Nrf2, a key transcription factor that mediates the induction of cellular antioxidant defense mechanisms, played an important protective role in limiting SCI induced cerebral inflammatory response and secondary neural injury [6, 7].
123
W. Jin et al.
However, it is not known whether the factor Nrf2 can be a therapeutic target in protecting against the cerebral inflammation associated with SCI. A wide range of natural and synthetic small molecules with diverse chemical backgrounds have been shown to induce the Nrf2 activity. Among these Nrf2 inducers, curcumin, which is an active component of the rhizome, Curcuma longa, exerts powerful antioxidant potential [8, 9]. It has been documented that curcumin protects the animals and cell lines against acute toxicity and oxidative insult presumably through inducing the nuclear translocation of transcription factor Nrf2, which in turn regulates the expression of many cytoprotective proteins such as NAD(P)H:quinone oxidoreductase 1, glutathione-S-transferase, glucuronyltransferase, and heme oxygenase-1 [10, 11]. Mostly synthesized in glial cells, Nrf2 is conceptualized as a major antioxidant defender in the central nerve system [12]. The expression of antioxidant and phase 2 detoxification enzyme genes is mediated through cis-acting sequences known as antioxidant response elements (AREs). Thus, activation of ARE is dependent on the translocation of Nrf2 from the cytoplasm to the nucleus [13]. However, to our knowledge, the therapeutic effect of the activation of Nrf2 by curcumin on the SCI induced inflammatory neural damage has not been studied. The purpose of this study was, therefore, to investigate the influence of curcumin administration in modulating SCIinduced up-regulation of inflammatory agents in the SCI.
Materials and methods Rats model of SCI The protocols of animal use and care conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health, and were approved by the Animal Care and Use Committee of Nanjing University. Male Sprague–Dawley rats (250–300 g) were purchased from the Animal Center of the Chinese Academy of Sciences, Shanghai, China. The rats were housed under temperature-controlled conditions with a 12-h light/dark cycle and ad libitum access to water and food. Following intraperitoneal anesthesia with sodium pentobarbital (50 mg/kg), a 2 cm midline incision was made along the vertebrae T7–T10. The thoracolumbar fascia and paraspinal musculature were incised along the spinous processes and retracted. A T8–T9 laminectomy was performed using an operating microscope. Thirty-seconds extradural compression with a vascular clip (30 g forces, Kent Scientific Corporation, INS 14120, USA) was performed around the exposed spinal cord to induce compression injury [14]. Then, the spinal cord was
123
irrigated with saline, while the muscles and skin were sutured. The site of the lesion was marked with a nondegradable suture. After operation procedures, the rats were returned to their cages under a heating lamp. Heart rate, arterial blood pressure and rectal temperature were monitored, and the rectal temperature was kept at 37 ± 0.5 °C throughout the experimental and recovery periods. The experimental groups consisted of a sham group, the SCI group, and the SCI ? curcumin group (n = 16 per group). Rats of the SCI ? curcumin group received intraperitoneal injections of curcumin at 100 mg/kg in PBS including 1 % DMSO at 15 min after SCI. The dose of curcumin used in this study was based on previous experiments [9]. Rats of sham and SCI groups received equal volume PBS including 1 % DMSO at 15 min after the surgery. At 72 h following sham or injury, rats were sacrificed for sample collection by cervical dislocation, after being tested for locomotion deficits (n = 16). For the electrophoretic mobility shift assay (EMSA), enzymelinked immunosorbent assay (ELISA), and wet/dry weight ratio, eight rats in each group were exsanguinated by cardiac puncture. The spinal cords spanning the injury site about 0.8 cm were rapidly excised. A portion of the tissue was harvested to determine wet/dry weight ratio detection. The remainder was immediately stored in liquid nitrogen until analysis. For terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) analysis, the other eight rats in each group were perfused via left ventricular puncture with cold saline (4 °C), followed by 4 % neutral-buffered formalin. The injured spinal cords about 0.8 cm were excised, stored overnight in 4 % neutralbuffered formalin, and then embedded in paraffin. Basso, Beattie, and Bresnahan (BBB) rating The hindlimb locomotion function of rats after SCI was scored in an open field according to the BBB locomotion rating scale of 0 (complete paralysis) to 21 (normal locomotion) as previously described [14]. The scale grossly assessed hindlimb movements, body weight support, forelimb-hindlimb coordination, and whole body movements. Each session was conducted for 5 min by two independent observers blinded to the experiments at 72 h after sham or injury. Wet/dry weight ratio assay Spinal cord water content was determined using the wet/ dry weight method as previously described [15]. Spinal cord samples were taken, and immediately weighed to obtain the wet weight (WW). The samples were dried in an oven for 24 h at 110 °C and weighed again to obtain the
Pathophysiology of traumatic spinal cord injury
dry weight (DW). Water content was calculated as [(WW - DW) 9 100]/WW. TUNEL study The formalin-fixed, paraffin-embedded tissue samples were sectioned at 4 lm thickness with a microtome. The sections (three sections per sample) were scored for apoptotic cells by the TUNEL method. The procedures were according to instructions of the kit (ISCDD, Boehringer Mannheim, Germany) and our laboratory methods [15]. Microscopy of the stained tissue sections was performed by a pathologist blinded to the experimental conditions. TUNEL-positive cells were counted in the section through the injury epicenter. The extent of spinal cord damage was evaluated by the apoptotic index (AI) which was the average percentage of TUNEL-positive cells in each section counted in ten cortical microscopic fields (at 2009 magnification).
Fig. 1 BBB locomotion assessment in the sham group, the SCI group and the SCI ? curcumin group (n = 16 per group). The figure shows that the BBB locomotion score was significantly decreased after SCI and could be up-regulated when treated with curcumin. **P \ 0.01 versus sham group; ##P \ 0.01 versus SCI group
Nuclear protein extract and EMSA Nrf2 and NF-jB DNA binding activities were determined by EMSA. Nuclear protein of spinal cord tissue was extracted and quantified as described previously [16]. EMSA was performed using a commercial kit (Gel Shift Assay System; Promega, Madison, WI) following the methods in our laboratory. Consensus oligonucleotide probes for Nrf2 (50 -TGG GGA ACC TGT GCT GAG TCA CTG GAG-30 ) and NF-jB (50 -AGT TGA GGG GAC TTT CCC AGG C-30 ) were end-labeled with [c-32P]ATP (Free Biotech., Beijing, China) with T4-polynucleotide kinase. EMSA was performed according to our previous study [16]. Levels of Nrf2 and NF-jB activity were quantified by computer-assisted optical densitometric analysis after three determinations for each band. ELISA analysis Spinal cord concentrations of inflammatory cytokines including TNF-a, IL-1b and IL-6 were quantified using ELISA kits specific for rats according to the manufacturers’ instructions (TNF-a from Diaclone Research, France; IL-1b, IL-6 from Biosource Europe SA, Belgium) and our previous study [17]. The cytokine contents were expressed as pg/mg protein.
Fig. 2 Spinal cord water content in sham group, SCI group and SCI ? curcumin group (n = 8 per group). Curcumin treatment significantly attenuated SCI-induced spinal cord edema in rats. **P \ 0.01 versus sham group; ##P \ 0.01 versus SCI group
experimental groups were determined by Fisher’s LSD posttest. Significance was assigned at P \ 0.05.
Results Hindlimb locomotion function
Statistical analysis Software SPSS 15.0 was used for the statistical analysis. All data were expressed as mean ± SD. The measurements were subjected to one-way ANOVA. Differences between
The BBB locomotion rating scale was developed based upon natural progression of locomotion recovery in rats with thoracic SCI. As shown in Fig. 1, the BBB score was approximately 21 in the sham group. Immediately after
123
W. Jin et al.
Fig. 3 TUNEL immunohistochemistry staining in the spinal cord tissue in the sham group, the SCI group and the SCI ? curcumin group (n = 8 per group). a sham group rats showing few TUNEL positive cells; b SCI group rats showing more TUNEL positive cells;
c SCI ? curcumin group rats showing less TUNEL positive cells than the SCI group; d Curcumin treatment significantly decreased the AI in the injured spinal cord following SCI. Scale bar length = 100 lm; **P \ 0.01 versus sham group; ##P \ 0.01 versus SCI group
surgery, the BBB score of every rat was approximately 0, which meant that the SCI model was successful. At 72 h after SCI, a decreased BBB score, which reflected an impairment of locomotion function, was observed in the SCI group in comparison with the sham group. In the SCI ? curcumin group, when compared with the SCI group, the BBB score was significantly increased.
increased compared with those in the sham group animals. In the SCI ? curcumin group, the AI in the spinal cord tissue was significantly decreased.
Spinal cord edema We investigated the spinal cord water content using the wet/dry weight ratio method. The water content was low in rats of the sham group as shown in Fig. 2. Significant increase in water content was detected in the spinal cord samples at 72 h after SCI when compared with rats in the sham group. The mean value of spinal cord water content was decreased by curcumin administration as compared with the SCI group.
Nrf2 and NF-jB DNA binding activity To determine the influence of curcumin on Nrf2 and NFjB activity in the spinal cord post-SCI, EMSA was performed to detect the DNA binding activity of Nrf2 and NFjB as described in ‘‘Materials and methods’’ (Fig. 4). Low Nrf2 and NF-jB DNA binding activity was detected in the sham group. At 72 h after SCI, both Nrf2 and NF-jB activity was significantly increased in the CI group in comparison with the sham group. After curcumin administration, the Nrf2 activity was markedly up-regulated but the NF-jB binding activity was significantly down-regulated in animals of the SCI ? curcumin group. Inflammatory cytokine concentrations
Spinal cord apoptosis Low AI was found in the sham group rats’ spinal cord tissue, as shown in Fig. 3. In the SCI group, the AI in the studied rat spinal cord tissue was found to be significantly
123
Concentrations of inflammatory cytokines including TNF-a, IL-1b and IL-6 were low in the rat spinal cords of the sham group, as shown in Fig. 5. Compared with the sham group, spinal cord levels of TNF-a, IL-1b and IL-6
Pathophysiology of traumatic spinal cord injury
Fig. 5 Concentrations of inflammatory cytokines in the spinal cord in the sham group, the SCI group and the SCI ? curcumin group (n = 8 per group). The figure indicates that concentrations of TNF-a, IL-1b and IL-6 in the injured spinal cord were significantly increased after SCI and could be suppressed when treated with curcumin. **P \ 0.01 versus sham group; ##P \ 0.01 versus SCI group
Fig. 4 Nrf2 and NF-jB DNA binding activities in the spinal cord in the sham group, the SCI group and the SCI group (n = 8 per group). EMSA autoradiography of Nrf2 and NF-jB DNA binding is shown on top of the graph, and the order of individual bands correspond to that of the graph bar. Curcumin treatment significantly up-regulated the Nrf2 activation but down-regulated the NF-jB activation in the injured spinal cord following SCI. **P \ 0.01 versus sham group; ## P \ 0.01 versus SCI group
were increased in the SCI group. In the SCI ? curcumin group, curcumin administration could lead to significantly decreased TNF-a, IL-1b and IL-6 concentrations in rats spinal cord tissue.
Discussion The main findings of this study were: (1) after curcumin administration, the SCI-induced locomotion deficit, spinal cord edema and apoptotic cell death were ameliorated; (2) the Nrf2 activity was significantly activated following SCI and could be further induced when treated with curcumin; (3) the inflammatory-related factors including NF-jB and proinflammatory cytokines in the injured spinal cord were
significantly up-regulated following SCI and could be suppressed when treated with curcumin. These findings reported here suggest for the first time that induction of the Nrf2 activity by curcumin can attenuate the spinal cord inflammation and reduce the neural damage following SCI. The BBB locomotion rating scale is a sensitive, valid, and reliable measure of locomotor function for rats [14]. Substantial evidence indicates that the grade of locomotor function deficit, the level of spinal cord edema, and the amount of apoptotic cell death correlates with the severity of SCI induced secondary neural injury [7, 18]. However, there is still no effective treatment for the secondary damage caused by SCI. Our previous studies illustrated the protective role of Nrf2 in the locomotor function deficit, spinal cord edema, and apoptotic cell death which played important roles and were the major part of the secondary neural injury following SCI [7]. In this current research, our data extended previous studies to show that induction of the Nrf2 activity by curcumin could reduce the locomotor function deficit, spinal cord edema, and apoptotic cell death in this SCI model. These findings suggested that curcumin could afford protection to the SCI-induced secondary neural injury. There have been several studies focusing on the neuroprotective effects of curcumin in SCI. As mentioned by Cemil et al. [19] in their literature, administration of curcumin provided neuroprotection in an experimental rat model of SCI. Their results suggested that these effects were accompanied by a decreased level of tissue
123
W. Jin et al.
malondialdehyde and increased levels of tissue glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase activity after SCI. Another research study indicated that treatment of SCI with curcumin inhibited apoptosis and neuron loss, quenched astrocyte activation, and significantly improved neurologic deficit in rats [20]. In a recent study by Ormond et al. [21], postinjury curcumin administration resulted in improved motor function recovery from SCI, correlating with no adverse effects noted in experimental animals. In this current research, our data are consistent with the previous studies. However, despite the demonstrated mechanisms of curcumin in neuroprotection, none of the previous studies have focused on the Nrf2 activity and spinal cord inflammation in relation to cytotoxic damage after SCI. To defend against exogenous toxins or injury, cells possess a large number of cytoprotective and detoxifying enzymes whose expression are rapidly increased in response to insults. Many of these genes contain a common promoter element called the ARE. Several transcription factors can bind to ARE, however, Nrf2 is the major one that binds to and activates the expression of these ARE-mediated gene products [22, 23]. Under basal conditions, Nrf2 is sequestered in the cytoplasm by the cytosolic regulatory protein Keap1. In conditions of toxins or injury, Nrf2 translocates from the cytoplasm to the nucleus [13, 24]. In recent research, the central role of Nrf2 in cell survival has been well established in vitro and in vivo [25, 26]. Our previous studies demonstrated that Nrf2 was activated, and protected mice against both oxidant and inflammatory effects after SCI [6, 7]. These provide the evidence that Nrf2 plays an important role on the secondary neural injury after SCI. Up-regulation Nrf2 may have great benefit for the SCI-induced spinal cord damage. A wide range of natural and synthetic small molecules with diverse chemical backgrounds are potent inducers of Nrf2 activity. Among them, curcumin, which has been used for centuries in traditional medicine, is of particular interest [9, 27]. In the previous research regarding curcumin and the Nrf2 signaling pathway, Carmona-Ramirez et al. [28] investigated the influence of curcumin on the activation of Nrf2 in a neurodegenerative model induced by quinolinic acid in rats. They put forward that neuroprotection induced by curcumin is associated with its ability to activate the Nrf2 cytoprotective pathway and to increase the total SOD and GSH-Px activities. In another study mentioned by Zhao et al. [29], curcumin administration in the cultured LO2 hepatocytes reduced ROS-mediated insulin resistance in hepatocytes through activation of the Nrf2 cytoprotective pathway. In our present study, we reported for the first time that, in the SCI model, the Nrf2 activity in the injured
123
spinal cord was significantly activated and could be promoted after curcumin administration, which is in remarkable agreement with the previous studies from other in vivo and vitro models. These results provide evidence that activation of Nrf2 by administration of tBHQ is a potential mechanism for its neuroprotection after SCI. There is converging evidence that the inflammatory response contributes to SCI progression. Activation of the NF-jB signaling pathway has been shown to be central to the pathophysiology of SCI-induced spinal cord inflammation [30]. NF-jB is one of the most important proinflammatory modulators, which can be activated by lesion-induced oxidative stress, bacterial endotoxin, or cytokines [31]. The functional importance of NF-jB in inflammation is based on its ability to regulate the promoters of multiple inflammatory cytokines including interleukins (ILs) and TNF-a [32]. NF-jB activation enhances the transcription of inflammatory cytokines, and the cytokines are known to in turn activate NF-jB. The positive feedback is believed to serve to amplify inflammatory signals and exacerbate neural damage after SCI. Our previous study demonstrated that Nrf2 played an important protective role in limiting the upregulation of NF-jB activity and pro-inflammatory cytokines after SCI [7]. In the present study, we extended these findings to show that induction of the Nrf2 activity by curcumin markedly decreased SCI-induced NF-jB activation and inflammatory cytokines production in the spinal cord. These results illustrated that curcumin administration could suppress the spinal cord inflammation following SCI. Although the precise mechanism regarding the antiinflammatory ability of curcumin remains elusive, several lines of evidence indicate that Nrf2 and NF-jB signaling pathways contribute to the pathophysiological process. The prevailing theory has been that Nrf2 interferes with inflammatory signaling pathways by inhibiting NF-jB activation through the maintenance of cellular redox status [33]. Curcumin, therefore, might play an important role in anti-inflammation by a mechanism of the augmentation of cellular antioxidative system via up-regulation of the Nrf2 signaling pathway resulting in decreased pro-inflammatory cytokines production via inactivation of the NF-jB signaling pathway. It is assured that further ingenious research is needed and will be conducted in our laboratory. Finally, in conclusion, to the best of our knowledge, this was the first study that elucidated the protective effect of curcumin on spinal cord inflammatory response and secondary neural injury following SCI. Our results suggested that the therapeutic benefit of curcumin might be due to its salutary effect on inducing the Nrf2 signaling pathway.
Pathophysiology of traumatic spinal cord injury Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (81200938). We thank Dr. Li-zhi Xu for technical assistance.
References 1. Choo AM, Liu J, Dvorak M, Tetzlaff W, Oxland TR. Secondary pathology following contusion, dislocation, and distraction spinal cord injuries. Exp Neurol. 2008;212:490–506. 2. Donnelly DJ, Popovich PG. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Exp Neurol. 2008;209:378–88. 3. Trivedi A, Olivas AD, Noble-Haeusslein LJ. Inflammation and spinal cord injury: infiltrating leukocytes as determinants of injury and repair processes. Clin Neurosci Res. 2006;6:283–92. 4. Rafati DS, Geissler K, Johnson K, Unabia G, Hulsebosch C, Nesic-Taylor O, Perez-Polo JR. Nuclear factor-kappaB decoy amelioration of spinal cord injury-induced inflammation and behavior outcomes. J Neurosci Res. 2008;86:566–80. 5. Chen X, Zhou C, Guo J, Sun K, Zhao N, Yang J, Sun Y, Liu X, Hibi T, Liu Z, Han J. Effects of dihydroxylphenyl lactic acid on inflammatory responses in spinal cord injury. Brain Res. 2011;1372:160–8. 6. Wang X, de Rivero Vaccari JP, Wang H, Diaz P, German R, Marcillo AE, Keane RW. Activation of the nuclear factor E2-related factor 2/antioxidant response element pathway is neuroprotective after spinal cord injury. J Neurotrauma. 2012;29:936–45. 7. Mao L, Wang H, Wang X, Liao H, Zhao X. Transcription factor Nrf2 protects the spinal cord from inflammation produced by spinal cord injury. J Surg Res. 2011;170:e105–15. 8. Farombi EO, Shrotriya S, Na HK, Kim SH, Surh YJ. Curcumin attenuates dimethylnitrosamine-induced liver injury in rats through Nrf2-mediated induction of heme oxygenase-1. Food Chem Toxicol. 2008;46:1279–87. 9. Yang C, Zhang X, Fan H, Liu Y. Curcumin upregulates transcription factor Nrf2, HO-1 expression and protects rat brains against focal ischemia. Brain Res. 2009;1282:133–41. 10. Tapia E, Soto V, Ortiz-Vega KM, Zarco-Ma´rquez G, MolinaJijo´n E, Cristo´bal-Garcı´a M, Santamarı´a J, Garcı´a-Nin˜o WR, Correa F, Zazueta C, Pedraza-Chaverri J. Curcumin induces Nrf2 nuclear translocation and prevents glomerular hypertension, hyperfiltration, oxidant stress, and the decrease in antioxidant enzymes in 5/6 nephrectomized rats. Oxid Med Cell Longev. 2012;2012:269039. 11. Sahin K, Orhan C, Tuzcu Z, Tuzcu M, Sahin N. Curcumin ameliorates heat stress via inhibition of oxidative stress and modulation of Nrf2/HO-1 pathway in quail. Food Chem Toxicol. 2012;50:4035–41. 12. Vargas MR, Johnson DA, Sirkis DW, Messing A, Johnson JA. Nrf2 activation in astrocytes protects against neurodegeneration in mouse models of familial amyotrophic lateral sclerosis. J Neurosci. 2008;28:13574–81. 13. Osburn WO, Wakabayashi N, Misra V, Nilles T, Biswal S, Trush MA, Kensler TW. Nrf2 regulates an adaptive response protecting against oxidative damage following diquat-mediated formation of superoxide anion. Arch Biochem Biophys. 2006;454:7–15. 14. Liang WB, Han QQ, Jin W, Xiao ZF, Huang JC, Ni HB, Chen B, Kong J, Wu J, Dai JW. The promotion of neurological recovery in the rat spinal cord crushed injury model by collagen-binding BDNF. Biomaterials. 2010;31:8634–41. 15. Jin W, Kong J, Lu TY, Wang HD, Ni HB, Wu J, Dai YX, Jiang J, Liang WB. Erythropoietin prevents secondary brain injury induced by cortical lesion in mice: possible involvement of Nrf2 signaling pathway. Ann Clin Lab Sci. 2011;41:25–32.
16. Jin W, Wang HD, Yan W, Zhu L, Hu ZG, Ding YS, Tang K. Role of Nrf2 in protection against traumatic brain injury in mice. J Neurotrauma. 2009;26:131–9. 17. Jin W, Wang HD, Yan W, Xu LZ, Wang XL, Zhao XN, Yang XH, Chen G, Ji Y. Disruption of Nrf2 enhances upregulation of nuclear factor-kappaB activity, proinflammatory cytokines, and intercellular adhesion molecule-1 in the brain after traumatic brain injury. Mediat Inflamm. 2008;2008:725174. 18. Sharma HS, Badgaiyan RD, Alm P, Mohanty S, Wiklund L. Neuroprotective effects of nitric oxide synthase inhibitors in spinal cord injury-induced pathophysiology and motor functions: an experimental study in the rat. Ann N Y Acad Sci. 2005;1053:422–34. 19. Cemil B, Topuz K, Demircan MN, Kurt G, Tun K, Kutlay M, Ipcioglu O, Kucukodaci Z. Curcumin improves early functional results after experimental spinal cord injury. Acta Neurochir (Wien). 2010;152:1583–90. 20. Lin MS, Lee YH, Chiu WT, Hung KS. Curcumin provides neuroprotection after spinal cord injury. J Surg Res. 2011;166:280–9. 21. Ormond DR, Peng H, Zeman R, Das K, Murali R, JhanwarUniyal M. Recovery from spinal cord injury using naturally occurring antiinflammatory compound curcumin. J Neurosurg Spine. 2012;16:497–503. 22. Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun. 1997;236:313–22. 23. Motohashi H, Yamamoto M. Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol Med. 2004;10:549–57. 24. Jain AK, Bloom DA, Jaiswal AK. Nuclear import and export signals in control of Nrf2. J Biol Chem. 2005;280:29158–68. 25. Lee JM, Calkins MJ, Chan K, Kan YW, Johnson JA. Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis. J Biol Chem. 2003;278:12029–38. 26. Calkins MJ, Jakel RJ, Johnson DA, Chan K, Kan YW, Johnson JA. Protection from mitochondrial complex II inhibition in vitro and in vivo by Nrf2-mediated transcription. Proc Natl Acad Sci USA. 2005;102:244–9. 27. Jiang H, Tian X, Guo Y, Duan W, Bu H, Li C. Activation of nuclear factor erythroid 2-related factor 2 cytoprotective signaling by curcumin protect primary spinal cord astrocytes against oxidative toxicity. Biol Pharm Bull. 2011;34:1194–7. 28. Carmona-Ramı´rez I, Santamarı´a A, Tobo´n-Velasco JC, OrozcoIbarra M, Gonza´lez-Herrera IG, Pedraza-Chaverrı´ J, Maldonado PD. Curcumin restores Nrf2 levels and prevents quinolinic acidinduced neurotoxicity. J Nutr Biochem. 2013;24:14–24. 29. Zhao SG, Li Q, Liu ZX, Wang JJ, Wang XX, Qin M, Wen QS. Curcumin attenuates insulin resistance in hepatocytes by inducing Nrf2 nuclear translocation. Hepatogastroenterology. 2011;58: 2106–11. 30. Bethea JR, Castro M, Keane RW, Lee TT, Dietrich WD, Yezierski RP. Traumatic spinal cord injury induces nuclear factorkappaB activation. J Neurosci. 1998;18:3251–60. 31. Chen F, Castranova V, Shi X, Dwmers LM. New insights into the role of nuclear factor-kappaB, a ubiquitous transcription factor in the initiation of diseases. Clin Chem. 1999;45:7–17. 32. Moon DO, Kim MO, Kang SH, Choi YH, Kim GY. Sulforaphane suppresses TNF-alpha-mediated activation of NF-kappaB and induces apoptosis through activation of reactive oxygen speciesdependent caspase-3. Cancer Lett. 2009;274:132–42. 33. Thimmulappa RK, Lee H, Rangasamy T, Reddy SP, Yamamoto M, Kensler TW, Biswal S. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J Clin Invest. 2006;116:984–95.
123
Inflammation Research
ORIGINAL RESEARCH PAPER
Anti-inflammatory effects of curcumin in experimental spinal cord injury in rats Wei Jin • Jing Wang • Tiansheng Zhu • Baoyu Yuan • Hongbin Ni Jian Jiang • Handong Wang • Weibang Liang
•
Received: 4 October 2013 / Revised: 17 December 2013 / Accepted: 12 January 2014 Ó Springer Basel 2014
Abstract Aim Antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) has been shown in our previous studies to play an important role in protection against spinal cord injury (SCI) induced inflammatory response. The objective of this study was to test whether curcumin, a novel Nrf2 activator, can protect the spinal cord against SCI-induced inflammatory damage. Methods Adult male Sprague–Dawley rats were subjected to laminectomy at T8–T9 and compression with a vascular clip. The spinal cords spanning the injury site about 0.8 cm were collected for testing. There were three groups: (a) sham group; (b) SCI group; and (c) SCI ? curcumin group. We measured Nrf2 and nuclear factor kappa B (NF-jB) binding activities by electrophoretic mobility shift assay, concentrations of tumor necrosis factor-a, interleukin-1b and interleukin-6 by enzymelinked immunosorbent assay, hindlimb locomotion function by Basso, Beattie, and Bresnahan rating, spinal cord edema by the wet/dry weight method, and apoptosis by
Responsible Editor: Ji Zhang. W. Jin and J. Wang contributed equally to this work. W. Jin J. Wang T. Zhu B. Yuan H. Ni J. Jiang W. Liang (&) Department of Neurosurgery, Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu, People’s Republic of China e-mail: [email protected] H. Wang (&) Department of Neurosurgery, Jinling Hospital, Medical School of Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu, People’s Republic of China e-mail: [email protected]
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis. Results Induction of the Nrf2 activity by curcumin markedly decreased NF-jB activation and inflammatory cytokines production in the injured spinal cord. Administration of curcumin also significantly ameliorated the secondary spinal cord damage, as shown by decreased severity of locomotion deficit, spinal cord edema, and apoptosis. Conclusion Post-SCI curcumin administration attenuates the inflammatory response in the injured spinal cord, and this may be a mechanism whereby curcumin improves the outcome following SCI. Keywords Curcumin Spinal cord injury Nrf2 Inflammatory response
Introduction Spinal cord injury (SCI) initiates a series of cellular and molecular cascade events, and a combination of secondary injury factors leads to a progressive neural injury [1]. Among these secondary injury factors, inflammatory response is the major one and plays an important role in regulating the pathogenesis of SCI [2, 3]. Increased levels of inflammation-related factors with the injured spinal cord, including tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), interleukin-6 (IL-6), and their mediator NF-jB, are believed to contribute to the neural damage [4, 5]. Previous studies of our laboratory demonstrated that Nrf2, a key transcription factor that mediates the induction of cellular antioxidant defense mechanisms, played an important protective role in limiting SCI induced cerebral inflammatory response and secondary neural injury [6, 7].
123
W. Jin et al.
However, it is not known whether the factor Nrf2 can be a therapeutic target in protecting against the cerebral inflammation associated with SCI. A wide range of natural and synthetic small molecules with diverse chemical backgrounds have been shown to induce the Nrf2 activity. Among these Nrf2 inducers, curcumin, which is an active component of the rhizome, Curcuma longa, exerts powerful antioxidant potential [8, 9]. It has been documented that curcumin protects the animals and cell lines against acute toxicity and oxidative insult presumably through inducing the nuclear translocation of transcription factor Nrf2, which in turn regulates the expression of many cytoprotective proteins such as NAD(P)H:quinone oxidoreductase 1, glutathione-S-transferase, glucuronyltransferase, and heme oxygenase-1 [10, 11]. Mostly synthesized in glial cells, Nrf2 is conceptualized as a major antioxidant defender in the central nerve system [12]. The expression of antioxidant and phase 2 detoxification enzyme genes is mediated through cis-acting sequences known as antioxidant response elements (AREs). Thus, activation of ARE is dependent on the translocation of Nrf2 from the cytoplasm to the nucleus [13]. However, to our knowledge, the therapeutic effect of the activation of Nrf2 by curcumin on the SCI induced inflammatory neural damage has not been studied. The purpose of this study was, therefore, to investigate the influence of curcumin administration in modulating SCIinduced up-regulation of inflammatory agents in the SCI.
Materials and methods Rats model of SCI The protocols of animal use and care conformed to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health, and were approved by the Animal Care and Use Committee of Nanjing University. Male Sprague–Dawley rats (250–300 g) were purchased from the Animal Center of the Chinese Academy of Sciences, Shanghai, China. The rats were housed under temperature-controlled conditions with a 12-h light/dark cycle and ad libitum access to water and food. Following intraperitoneal anesthesia with sodium pentobarbital (50 mg/kg), a 2 cm midline incision was made along the vertebrae T7–T10. The thoracolumbar fascia and paraspinal musculature were incised along the spinous processes and retracted. A T8–T9 laminectomy was performed using an operating microscope. Thirty-seconds extradural compression with a vascular clip (30 g forces, Kent Scientific Corporation, INS 14120, USA) was performed around the exposed spinal cord to induce compression injury [14]. Then, the spinal cord was
123
irrigated with saline, while the muscles and skin were sutured. The site of the lesion was marked with a nondegradable suture. After operation procedures, the rats were returned to their cages under a heating lamp. Heart rate, arterial blood pressure and rectal temperature were monitored, and the rectal temperature was kept at 37 ± 0.5 °C throughout the experimental and recovery periods. The experimental groups consisted of a sham group, the SCI group, and the SCI ? curcumin group (n = 16 per group). Rats of the SCI ? curcumin group received intraperitoneal injections of curcumin at 100 mg/kg in PBS including 1 % DMSO at 15 min after SCI. The dose of curcumin used in this study was based on previous experiments [9]. Rats of sham and SCI groups received equal volume PBS including 1 % DMSO at 15 min after the surgery. At 72 h following sham or injury, rats were sacrificed for sample collection by cervical dislocation, after being tested for locomotion deficits (n = 16). For the electrophoretic mobility shift assay (EMSA), enzymelinked immunosorbent assay (ELISA), and wet/dry weight ratio, eight rats in each group were exsanguinated by cardiac puncture. The spinal cords spanning the injury site about 0.8 cm were rapidly excised. A portion of the tissue was harvested to determine wet/dry weight ratio detection. The remainder was immediately stored in liquid nitrogen until analysis. For terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) analysis, the other eight rats in each group were perfused via left ventricular puncture with cold saline (4 °C), followed by 4 % neutral-buffered formalin. The injured spinal cords about 0.8 cm were excised, stored overnight in 4 % neutralbuffered formalin, and then embedded in paraffin. Basso, Beattie, and Bresnahan (BBB) rating The hindlimb locomotion function of rats after SCI was scored in an open field according to the BBB locomotion rating scale of 0 (complete paralysis) to 21 (normal locomotion) as previously described [14]. The scale grossly assessed hindlimb movements, body weight support, forelimb-hindlimb coordination, and whole body movements. Each session was conducted for 5 min by two independent observers blinded to the experiments at 72 h after sham or injury. Wet/dry weight ratio assay Spinal cord water content was determined using the wet/ dry weight method as previously described [15]. Spinal cord samples were taken, and immediately weighed to obtain the wet weight (WW). The samples were dried in an oven for 24 h at 110 °C and weighed again to obtain the
Pathophysiology of traumatic spinal cord injury
dry weight (DW). Water content was calculated as [(WW - DW) 9 100]/WW. TUNEL study The formalin-fixed, paraffin-embedded tissue samples were sectioned at 4 lm thickness with a microtome. The sections (three sections per sample) were scored for apoptotic cells by the TUNEL method. The procedures were according to instructions of the kit (ISCDD, Boehringer Mannheim, Germany) and our laboratory methods [15]. Microscopy of the stained tissue sections was performed by a pathologist blinded to the experimental conditions. TUNEL-positive cells were counted in the section through the injury epicenter. The extent of spinal cord damage was evaluated by the apoptotic index (AI) which was the average percentage of TUNEL-positive cells in each section counted in ten cortical microscopic fields (at 2009 magnification).
Fig. 1 BBB locomotion assessment in the sham group, the SCI group and the SCI ? curcumin group (n = 16 per group). The figure shows that the BBB locomotion score was significantly decreased after SCI and could be up-regulated when treated with curcumin. **P \ 0.01 versus sham group; ##P \ 0.01 versus SCI group
Nuclear protein extract and EMSA Nrf2 and NF-jB DNA binding activities were determined by EMSA. Nuclear protein of spinal cord tissue was extracted and quantified as described previously [16]. EMSA was performed using a commercial kit (Gel Shift Assay System; Promega, Madison, WI) following the methods in our laboratory. Consensus oligonucleotide probes for Nrf2 (50 -TGG GGA ACC TGT GCT GAG TCA CTG GAG-30 ) and NF-jB (50 -AGT TGA GGG GAC TTT CCC AGG C-30 ) were end-labeled with [c-32P]ATP (Free Biotech., Beijing, China) with T4-polynucleotide kinase. EMSA was performed according to our previous study [16]. Levels of Nrf2 and NF-jB activity were quantified by computer-assisted optical densitometric analysis after three determinations for each band. ELISA analysis Spinal cord concentrations of inflammatory cytokines including TNF-a, IL-1b and IL-6 were quantified using ELISA kits specific for rats according to the manufacturers’ instructions (TNF-a from Diaclone Research, France; IL-1b, IL-6 from Biosource Europe SA, Belgium) and our previous study [17]. The cytokine contents were expressed as pg/mg protein.
Fig. 2 Spinal cord water content in sham group, SCI group and SCI ? curcumin group (n = 8 per group). Curcumin treatment significantly attenuated SCI-induced spinal cord edema in rats. **P \ 0.01 versus sham group; ##P \ 0.01 versus SCI group
experimental groups were determined by Fisher’s LSD posttest. Significance was assigned at P \ 0.05.
Results Hindlimb locomotion function
Statistical analysis Software SPSS 15.0 was used for the statistical analysis. All data were expressed as mean ± SD. The measurements were subjected to one-way ANOVA. Differences between
The BBB locomotion rating scale was developed based upon natural progression of locomotion recovery in rats with thoracic SCI. As shown in Fig. 1, the BBB score was approximately 21 in the sham group. Immediately after
123
W. Jin et al.
Fig. 3 TUNEL immunohistochemistry staining in the spinal cord tissue in the sham group, the SCI group and the SCI ? curcumin group (n = 8 per group). a sham group rats showing few TUNEL positive cells; b SCI group rats showing more TUNEL positive cells;
c SCI ? curcumin group rats showing less TUNEL positive cells than the SCI group; d Curcumin treatment significantly decreased the AI in the injured spinal cord following SCI. Scale bar length = 100 lm; **P \ 0.01 versus sham group; ##P \ 0.01 versus SCI group
surgery, the BBB score of every rat was approximately 0, which meant that the SCI model was successful. At 72 h after SCI, a decreased BBB score, which reflected an impairment of locomotion function, was observed in the SCI group in comparison with the sham group. In the SCI ? curcumin group, when compared with the SCI group, the BBB score was significantly increased.
increased compared with those in the sham group animals. In the SCI ? curcumin group, the AI in the spinal cord tissue was significantly decreased.
Spinal cord edema We investigated the spinal cord water content using the wet/dry weight ratio method. The water content was low in rats of the sham group as shown in Fig. 2. Significant increase in water content was detected in the spinal cord samples at 72 h after SCI when compared with rats in the sham group. The mean value of spinal cord water content was decreased by curcumin administration as compared with the SCI group.
Nrf2 and NF-jB DNA binding activity To determine the influence of curcumin on Nrf2 and NFjB activity in the spinal cord post-SCI, EMSA was performed to detect the DNA binding activity of Nrf2 and NFjB as described in ‘‘Materials and methods’’ (Fig. 4). Low Nrf2 and NF-jB DNA binding activity was detected in the sham group. At 72 h after SCI, both Nrf2 and NF-jB activity was significantly increased in the CI group in comparison with the sham group. After curcumin administration, the Nrf2 activity was markedly up-regulated but the NF-jB binding activity was significantly down-regulated in animals of the SCI ? curcumin group. Inflammatory cytokine concentrations
Spinal cord apoptosis Low AI was found in the sham group rats’ spinal cord tissue, as shown in Fig. 3. In the SCI group, the AI in the studied rat spinal cord tissue was found to be significantly
123
Concentrations of inflammatory cytokines including TNF-a, IL-1b and IL-6 were low in the rat spinal cords of the sham group, as shown in Fig. 5. Compared with the sham group, spinal cord levels of TNF-a, IL-1b and IL-6
Pathophysiology of traumatic spinal cord injury
Fig. 5 Concentrations of inflammatory cytokines in the spinal cord in the sham group, the SCI group and the SCI ? curcumin group (n = 8 per group). The figure indicates that concentrations of TNF-a, IL-1b and IL-6 in the injured spinal cord were significantly increased after SCI and could be suppressed when treated with curcumin. **P \ 0.01 versus sham group; ##P \ 0.01 versus SCI group
Fig. 4 Nrf2 and NF-jB DNA binding activities in the spinal cord in the sham group, the SCI group and the SCI group (n = 8 per group). EMSA autoradiography of Nrf2 and NF-jB DNA binding is shown on top of the graph, and the order of individual bands correspond to that of the graph bar. Curcumin treatment significantly up-regulated the Nrf2 activation but down-regulated the NF-jB activation in the injured spinal cord following SCI. **P \ 0.01 versus sham group; ## P \ 0.01 versus SCI group
were increased in the SCI group. In the SCI ? curcumin group, curcumin administration could lead to significantly decreased TNF-a, IL-1b and IL-6 concentrations in rats spinal cord tissue.
Discussion The main findings of this study were: (1) after curcumin administration, the SCI-induced locomotion deficit, spinal cord edema and apoptotic cell death were ameliorated; (2) the Nrf2 activity was significantly activated following SCI and could be further induced when treated with curcumin; (3) the inflammatory-related factors including NF-jB and proinflammatory cytokines in the injured spinal cord were
significantly up-regulated following SCI and could be suppressed when treated with curcumin. These findings reported here suggest for the first time that induction of the Nrf2 activity by curcumin can attenuate the spinal cord inflammation and reduce the neural damage following SCI. The BBB locomotion rating scale is a sensitive, valid, and reliable measure of locomotor function for rats [14]. Substantial evidence indicates that the grade of locomotor function deficit, the level of spinal cord edema, and the amount of apoptotic cell death correlates with the severity of SCI induced secondary neural injury [7, 18]. However, there is still no effective treatment for the secondary damage caused by SCI. Our previous studies illustrated the protective role of Nrf2 in the locomotor function deficit, spinal cord edema, and apoptotic cell death which played important roles and were the major part of the secondary neural injury following SCI [7]. In this current research, our data extended previous studies to show that induction of the Nrf2 activity by curcumin could reduce the locomotor function deficit, spinal cord edema, and apoptotic cell death in this SCI model. These findings suggested that curcumin could afford protection to the SCI-induced secondary neural injury. There have been several studies focusing on the neuroprotective effects of curcumin in SCI. As mentioned by Cemil et al. [19] in their literature, administration of curcumin provided neuroprotection in an experimental rat model of SCI. Their results suggested that these effects were accompanied by a decreased level of tissue
123
W. Jin et al.
malondialdehyde and increased levels of tissue glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase activity after SCI. Another research study indicated that treatment of SCI with curcumin inhibited apoptosis and neuron loss, quenched astrocyte activation, and significantly improved neurologic deficit in rats [20]. In a recent study by Ormond et al. [21], postinjury curcumin administration resulted in improved motor function recovery from SCI, correlating with no adverse effects noted in experimental animals. In this current research, our data are consistent with the previous studies. However, despite the demonstrated mechanisms of curcumin in neuroprotection, none of the previous studies have focused on the Nrf2 activity and spinal cord inflammation in relation to cytotoxic damage after SCI. To defend against exogenous toxins or injury, cells possess a large number of cytoprotective and detoxifying enzymes whose expression are rapidly increased in response to insults. Many of these genes contain a common promoter element called the ARE. Several transcription factors can bind to ARE, however, Nrf2 is the major one that binds to and activates the expression of these ARE-mediated gene products [22, 23]. Under basal conditions, Nrf2 is sequestered in the cytoplasm by the cytosolic regulatory protein Keap1. In conditions of toxins or injury, Nrf2 translocates from the cytoplasm to the nucleus [13, 24]. In recent research, the central role of Nrf2 in cell survival has been well established in vitro and in vivo [25, 26]. Our previous studies demonstrated that Nrf2 was activated, and protected mice against both oxidant and inflammatory effects after SCI [6, 7]. These provide the evidence that Nrf2 plays an important role on the secondary neural injury after SCI. Up-regulation Nrf2 may have great benefit for the SCI-induced spinal cord damage. A wide range of natural and synthetic small molecules with diverse chemical backgrounds are potent inducers of Nrf2 activity. Among them, curcumin, which has been used for centuries in traditional medicine, is of particular interest [9, 27]. In the previous research regarding curcumin and the Nrf2 signaling pathway, Carmona-Ramirez et al. [28] investigated the influence of curcumin on the activation of Nrf2 in a neurodegenerative model induced by quinolinic acid in rats. They put forward that neuroprotection induced by curcumin is associated with its ability to activate the Nrf2 cytoprotective pathway and to increase the total SOD and GSH-Px activities. In another study mentioned by Zhao et al. [29], curcumin administration in the cultured LO2 hepatocytes reduced ROS-mediated insulin resistance in hepatocytes through activation of the Nrf2 cytoprotective pathway. In our present study, we reported for the first time that, in the SCI model, the Nrf2 activity in the injured
123
spinal cord was significantly activated and could be promoted after curcumin administration, which is in remarkable agreement with the previous studies from other in vivo and vitro models. These results provide evidence that activation of Nrf2 by administration of tBHQ is a potential mechanism for its neuroprotection after SCI. There is converging evidence that the inflammatory response contributes to SCI progression. Activation of the NF-jB signaling pathway has been shown to be central to the pathophysiology of SCI-induced spinal cord inflammation [30]. NF-jB is one of the most important proinflammatory modulators, which can be activated by lesion-induced oxidative stress, bacterial endotoxin, or cytokines [31]. The functional importance of NF-jB in inflammation is based on its ability to regulate the promoters of multiple inflammatory cytokines including interleukins (ILs) and TNF-a [32]. NF-jB activation enhances the transcription of inflammatory cytokines, and the cytokines are known to in turn activate NF-jB. The positive feedback is believed to serve to amplify inflammatory signals and exacerbate neural damage after SCI. Our previous study demonstrated that Nrf2 played an important protective role in limiting the upregulation of NF-jB activity and pro-inflammatory cytokines after SCI [7]. In the present study, we extended these findings to show that induction of the Nrf2 activity by curcumin markedly decreased SCI-induced NF-jB activation and inflammatory cytokines production in the spinal cord. These results illustrated that curcumin administration could suppress the spinal cord inflammation following SCI. Although the precise mechanism regarding the antiinflammatory ability of curcumin remains elusive, several lines of evidence indicate that Nrf2 and NF-jB signaling pathways contribute to the pathophysiological process. The prevailing theory has been that Nrf2 interferes with inflammatory signaling pathways by inhibiting NF-jB activation through the maintenance of cellular redox status [33]. Curcumin, therefore, might play an important role in anti-inflammation by a mechanism of the augmentation of cellular antioxidative system via up-regulation of the Nrf2 signaling pathway resulting in decreased pro-inflammatory cytokines production via inactivation of the NF-jB signaling pathway. It is assured that further ingenious research is needed and will be conducted in our laboratory. Finally, in conclusion, to the best of our knowledge, this was the first study that elucidated the protective effect of curcumin on spinal cord inflammatory response and secondary neural injury following SCI. Our results suggested that the therapeutic benefit of curcumin might be due to its salutary effect on inducing the Nrf2 signaling pathway.
Pathophysiology of traumatic spinal cord injury Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (81200938). We thank Dr. Li-zhi Xu for technical assistance.
References 1. Choo AM, Liu J, Dvorak M, Tetzlaff W, Oxland TR. Secondary pathology following contusion, dislocation, and distraction spinal cord injuries. Exp Neurol. 2008;212:490–506. 2. Donnelly DJ, Popovich PG. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Exp Neurol. 2008;209:378–88. 3. Trivedi A, Olivas AD, Noble-Haeusslein LJ. Inflammation and spinal cord injury: infiltrating leukocytes as determinants of injury and repair processes. Clin Neurosci Res. 2006;6:283–92. 4. Rafati DS, Geissler K, Johnson K, Unabia G, Hulsebosch C, Nesic-Taylor O, Perez-Polo JR. Nuclear factor-kappaB decoy amelioration of spinal cord injury-induced inflammation and behavior outcomes. J Neurosci Res. 2008;86:566–80. 5. Chen X, Zhou C, Guo J, Sun K, Zhao N, Yang J, Sun Y, Liu X, Hibi T, Liu Z, Han J. Effects of dihydroxylphenyl lactic acid on inflammatory responses in spinal cord injury. Brain Res. 2011;1372:160–8. 6. Wang X, de Rivero Vaccari JP, Wang H, Diaz P, German R, Marcillo AE, Keane RW. Activation of the nuclear factor E2-related factor 2/antioxidant response element pathway is neuroprotective after spinal cord injury. J Neurotrauma. 2012;29:936–45. 7. Mao L, Wang H, Wang X, Liao H, Zhao X. Transcription factor Nrf2 protects the spinal cord from inflammation produced by spinal cord injury. J Surg Res. 2011;170:e105–15. 8. Farombi EO, Shrotriya S, Na HK, Kim SH, Surh YJ. Curcumin attenuates dimethylnitrosamine-induced liver injury in rats through Nrf2-mediated induction of heme oxygenase-1. Food Chem Toxicol. 2008;46:1279–87. 9. Yang C, Zhang X, Fan H, Liu Y. Curcumin upregulates transcription factor Nrf2, HO-1 expression and protects rat brains against focal ischemia. Brain Res. 2009;1282:133–41. 10. Tapia E, Soto V, Ortiz-Vega KM, Zarco-Ma´rquez G, MolinaJijo´n E, Cristo´bal-Garcı´a M, Santamarı´a J, Garcı´a-Nin˜o WR, Correa F, Zazueta C, Pedraza-Chaverri J. Curcumin induces Nrf2 nuclear translocation and prevents glomerular hypertension, hyperfiltration, oxidant stress, and the decrease in antioxidant enzymes in 5/6 nephrectomized rats. Oxid Med Cell Longev. 2012;2012:269039. 11. Sahin K, Orhan C, Tuzcu Z, Tuzcu M, Sahin N. Curcumin ameliorates heat stress via inhibition of oxidative stress and modulation of Nrf2/HO-1 pathway in quail. Food Chem Toxicol. 2012;50:4035–41. 12. Vargas MR, Johnson DA, Sirkis DW, Messing A, Johnson JA. Nrf2 activation in astrocytes protects against neurodegeneration in mouse models of familial amyotrophic lateral sclerosis. J Neurosci. 2008;28:13574–81. 13. Osburn WO, Wakabayashi N, Misra V, Nilles T, Biswal S, Trush MA, Kensler TW. Nrf2 regulates an adaptive response protecting against oxidative damage following diquat-mediated formation of superoxide anion. Arch Biochem Biophys. 2006;454:7–15. 14. Liang WB, Han QQ, Jin W, Xiao ZF, Huang JC, Ni HB, Chen B, Kong J, Wu J, Dai JW. The promotion of neurological recovery in the rat spinal cord crushed injury model by collagen-binding BDNF. Biomaterials. 2010;31:8634–41. 15. Jin W, Kong J, Lu TY, Wang HD, Ni HB, Wu J, Dai YX, Jiang J, Liang WB. Erythropoietin prevents secondary brain injury induced by cortical lesion in mice: possible involvement of Nrf2 signaling pathway. Ann Clin Lab Sci. 2011;41:25–32.
16. Jin W, Wang HD, Yan W, Zhu L, Hu ZG, Ding YS, Tang K. Role of Nrf2 in protection against traumatic brain injury in mice. J Neurotrauma. 2009;26:131–9. 17. Jin W, Wang HD, Yan W, Xu LZ, Wang XL, Zhao XN, Yang XH, Chen G, Ji Y. Disruption of Nrf2 enhances upregulation of nuclear factor-kappaB activity, proinflammatory cytokines, and intercellular adhesion molecule-1 in the brain after traumatic brain injury. Mediat Inflamm. 2008;2008:725174. 18. Sharma HS, Badgaiyan RD, Alm P, Mohanty S, Wiklund L. Neuroprotective effects of nitric oxide synthase inhibitors in spinal cord injury-induced pathophysiology and motor functions: an experimental study in the rat. Ann N Y Acad Sci. 2005;1053:422–34. 19. Cemil B, Topuz K, Demircan MN, Kurt G, Tun K, Kutlay M, Ipcioglu O, Kucukodaci Z. Curcumin improves early functional results after experimental spinal cord injury. Acta Neurochir (Wien). 2010;152:1583–90. 20. Lin MS, Lee YH, Chiu WT, Hung KS. Curcumin provides neuroprotection after spinal cord injury. J Surg Res. 2011;166:280–9. 21. Ormond DR, Peng H, Zeman R, Das K, Murali R, JhanwarUniyal M. Recovery from spinal cord injury using naturally occurring antiinflammatory compound curcumin. J Neurosurg Spine. 2012;16:497–503. 22. Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun. 1997;236:313–22. 23. Motohashi H, Yamamoto M. Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol Med. 2004;10:549–57. 24. Jain AK, Bloom DA, Jaiswal AK. Nuclear import and export signals in control of Nrf2. J Biol Chem. 2005;280:29158–68. 25. Lee JM, Calkins MJ, Chan K, Kan YW, Johnson JA. Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis. J Biol Chem. 2003;278:12029–38. 26. Calkins MJ, Jakel RJ, Johnson DA, Chan K, Kan YW, Johnson JA. Protection from mitochondrial complex II inhibition in vitro and in vivo by Nrf2-mediated transcription. Proc Natl Acad Sci USA. 2005;102:244–9. 27. Jiang H, Tian X, Guo Y, Duan W, Bu H, Li C. Activation of nuclear factor erythroid 2-related factor 2 cytoprotective signaling by curcumin protect primary spinal cord astrocytes against oxidative toxicity. Biol Pharm Bull. 2011;34:1194–7. 28. Carmona-Ramı´rez I, Santamarı´a A, Tobo´n-Velasco JC, OrozcoIbarra M, Gonza´lez-Herrera IG, Pedraza-Chaverrı´ J, Maldonado PD. Curcumin restores Nrf2 levels and prevents quinolinic acidinduced neurotoxicity. J Nutr Biochem. 2013;24:14–24. 29. Zhao SG, Li Q, Liu ZX, Wang JJ, Wang XX, Qin M, Wen QS. Curcumin attenuates insulin resistance in hepatocytes by inducing Nrf2 nuclear translocation. Hepatogastroenterology. 2011;58: 2106–11. 30. Bethea JR, Castro M, Keane RW, Lee TT, Dietrich WD, Yezierski RP. Traumatic spinal cord injury induces nuclear factorkappaB activation. J Neurosci. 1998;18:3251–60. 31. Chen F, Castranova V, Shi X, Dwmers LM. New insights into the role of nuclear factor-kappaB, a ubiquitous transcription factor in the initiation of diseases. Clin Chem. 1999;45:7–17. 32. Moon DO, Kim MO, Kang SH, Choi YH, Kim GY. Sulforaphane suppresses TNF-alpha-mediated activation of NF-kappaB and induces apoptosis through activation of reactive oxygen speciesdependent caspase-3. Cancer Lett. 2009;274:132–42. 33. Thimmulappa RK, Lee H, Rangasamy T, Reddy SP, Yamamoto M, Kensler TW, Biswal S. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J Clin Invest. 2006;116:984–95.
123
