Journal of the Neurological Sciences 346 (2014) 51–59
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Curcumin improves the integrity of blood–spinal cord barrier after compressive spinal cord injury in rats De-shui Yu a, Yang Cao a,⁎, Xi-fan Mei a, Yan-feng Wang b, Zhong-kai Fan a, Yan-song Wang a, Gang Lv a a b
Department of Orthopaedics, The First Affiliated Hospital, Liaoning Medical University, People Street No. 5-2, GuTa District, Jinzhou 121001, Liaoning Province, People's Republic of China Department of Orthopaedics, the First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, People's Republic of China
a r t i c l e
i n f o
Article history: Received 13 October 2013 Received in revised form 23 July 2014 Accepted 25 July 2014 Available online 4 August 2014 Keywords: Curcumin Spinal cord injury Blood–spinal cord barrier Tight junction Heme oxygenase-1 Tight junction protein
a b s t r a c t Previous studies have shown that curcumin (Cur) can produce potent neuroprotective effects against damage due to spinal cord injury (SCI). However, whether Cur can preserve the function of the blood–spinal cord barrier (BSCB) is unclear. The present study was performed to investigate the mechanism underlying BSCB permeability changes, which were induced by treatment with Cur (75, 150, and 300 mg/kg, i.p.) after compressive SCI in rats. BSCB permeability was evaluated by Evans blue leakage. Motor recovery of rats with SCI was assessed using the Basso, Beattie, and Bresnahan scoring system every day until the 21st days post-injury. The protein levels of heme oxygenase-1 (HO-1), tight junction protein, and inflammatory factors were analyzed by western blots. The expression of the inflammatory factors tumor necrosis factor-α (TNF-α) and nuclear factor-kappaB (NF-κB) mRNA was determined with reverse transcription-polymerase chain reactions. Treatment with Cur (150 and 300 mg/kg) significantly reduced Evans blue leakage into the spinal cord tissue at 24 h after SCI. Cur (150 mg/kg) significantly increased HO-1 protein expression. The levels of TNF-α and NF-κB mRNA and protein greatly increased at 24 h after SCI, and this increase was significantly attenuated by Cur treatment. ZO-1 and occludin expression was upregulated by Cur (150 mg/kg) treatment after SCI, and this effect was blocked by the HO-1 inhibitor zinc protoporphyrin. Long-term effects of Cur on motor recovery after SCI were observed. Our results indicated that Cur can improve motor function after SCI, which could correlate with improvements in BSCB integrity. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Spinal cord injury (SCI) is one of the most devastating traumatic conditions that induce primary and secondary tissue damage, resulting in postoperative complications, including spinal cord edema and cell death in the injured areas [1]. Recently, it has been reported that blood–spinal cord barrier (BSCB) disruption is associated with increased mortality after endovascular therapy, and improvements in the BSCB functioning can significantly reduce secondary nerve injury [2,3], suggesting that early repairs of the BSCB have important significance in the treatment of SCI. Therefore, drugs that reduce BSCB breakdown might improve outcomes of patients with SCI by limiting more aggressive surgical resections in confined spaces. Curcumin (Cur), which is a major active component of the food ingredient turmeric, is isolated from the powdered dry rhizome of Curcuma longa Linn. This component has a variety of pharmacological activities, including anti-oxidative, anti-inflammatory, anti-carcinogenic, and anti-HIV effects [4–6]. Previous studies have revealed that Cur exerts
⁎ Corresponding author. Tel./fax: +86 4164197717. E-mail address: [email protected] (Y. Cao).
http://dx.doi.org/10.1016/j.jns.2014.07.056 0022-510X/© 2014 Elsevier B.V. All rights reserved.
a neuroprotective role in diseases of the central nervous system, such as cerebral ischemia and traumatic brain injury [7,8]. Cur treatment has been shown to reduce edema and inflammation in the lesion areas of rats with SCI, suggesting its potential as a therapeutic agent [9]. However, the effects of Cur on the BSCB are still unclear. Recently, numerous studies have shown that the induction of heme oxygenase-1 (HO-1) is an important cellular protective mechanism against oxidative injury [10]. Due to the cumulative effects of HO-1 on heme catabolism and the generation of biologically active downstream products, HO-1 induction might serve as a protective mechanism against inflammatory injury. Inflammatory factors have been shown to increase BSCB permeability by timedependently modulating the expression and distribution of tight junction (TJ) proteins [11,12]. Cur, which is a naturally occurring potent inducer of HO-1 [13,14], provides protection against the release of inflammatory factors [15]. Recently, Cur has been demonstrated to protect human intestinal epithelial cells against the H2O2-induced disruption of TJ and barrier dysfunction through the HO-1 pathway [10]. Recently, Wang et al. have demonstrated that Cur preserves the permeability of the blood–brain barrier (BBB) during hypoxia by upregulating HO-1 expression in brain microvascular endothelial cells [16]. Therefore, we hypothesized that Cur may limit the SCI-induced disruption of TJs and barrier dysfunction through the HO-1 pathway. To test this hypothesis, we utilized a
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compressive SCI model in rat and evaluated the effects of Cur on HO-1, inflammatory factors, TJ disruption, BSCB dysfunction, and motor function that were induced by SCI. 2. Materials and methods 2.1. Establishment of spinal cord compression injury model in rats The adult male Sprang–Dawley rats weighing 210–260 g were purchased from the Experimental Animals Center of Liaoning Medical University. All animal experiments were conducted in accordance with the National Institute of Health Guide on the Care and Use of Laboratory Animals. The spinal cords were injured by arterial clamp with a closing force of 20 g (5 min) at thoracic level 8–10 (T8–10), using an established compression model. Rats were randomly and blindly divided into four groups, including sham-operated group, SCI group, SCI + Cur group and SCI + Cur +
ZnPP group. Sham-operated group, sham surgery included general anesthesia, skin incision, and laminectomy but clip compression was not applied; SCI group, rats were given a compression injury as the abovementioned method; SCI + Cur group, rats received immediately the treatment of Cur (Sigma-Aldrich) at the doses of 75, 150 and 300 mg/kg (i.p.) at 20 min after SCI and were given the administration of Cur every 24 h until the 3rd day after SCI, respectively; SCI + Cur + ZnPP group, rats were injected 25 μmol/kg of Znpp (i.p.) at 2 h before SCI and then were given the treatment of Cur (150 mg/kg, i.p.) every 24 h until the 3rd day after SCI. Six rats of each group were used in the following experiments respectively. 2.2. Measurement of BSCB permeability by Evans blue leakage BSCB permeability was quantitatively evaluated by extravasation of Evans blue (EB) as a marker of albumin extravasation. Briefly, rats for different time point group were injected 2% EB (2 ml/kg, iv). Two
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Fig. 1. The permeability of blood–spinal cord barrier was assessed by Evans blue extravasation (μg/g spinal cord tissue) and immunofluorescence at different time point in SCI group, SCI + Cur group (150 mg/kg, i.p.) and SCI + Cur + Znpp group. Data present means ± S.D. (n = 6, each). ⁎⁎P b 0.01 vs. SCI group. #P b 0.05 vs. SCI + Cur group.
D. Yu et al. / Journal of the Neurological Sciences 346 (2014) 51–59
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Fig. 1 (continued).
hours after EB injection, rats in different groups were transcardially perfused with 0.9% normal saline until colorless perfusion fluid was obtained from the right atrium. The injured spinal cord tissues were weighed and immersed in formamide (1 ml/100 mg) at 60 °C for 24 h. The optical density of the supernatant was examined with enzyme-labelled meter (at 620 nm, TECAN). The quantitative calculation of the dye content in the spinal cord tissue was based on the external standards dissolved in the same solvent. Rats for 1, 7 and 21 day post-SCI group were fixed by perfusion with 4% paraformaldehyde at 2 h after EB injection. The spinal cord tissues in different group were cut into 12 μm thickness at −27 °C using frozensection machine (Thermo Shandon). Evans blue leakage at the vascular level was also observed at day 1, day 7 and day 21 groups using immunofluorescence microscopy (IX71, Olympus).
2.3. Grading of motor disturbance Evaluation of motor dysfunction was graded using the modified murine Basso, Beattie, and Bresnahan (BBB) scoring system [17]. Motor function of rats that suffered from spinal cord compression injury was evaluated every day until the 21st day after SCI. 2.4. RT-PCR analysis The mRNA expression levels of tumor necrosis factor-α (TNF-α) and nuclear factor-kappaB (NF-κB) were determined by RT-PCR for 24 h groups post-SCI in rats. Total RNA was isolated from the injured spinal cord samples and purified using RNAout kit (Takara). Following the manufacturer's instructions, cDNAs were obtained from 1 μg of total
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RNA from each example. The oligonucleotide primers were as follows: NF-κB (P65) (381 bp): (forward) 5′CCT ATC CAC GAC AAC CTT GC 3′ and (reverse) 5′CAT AGA TGC TGC TGA CCC AAC 3′; TNF-α (413 bp): (forward) 5′CCC CAA GTG ACA AGC CAG TA 3′ and (reverse) 5′CAA AGT CCA GAT TAG GCA GAT 3′; β-actin (493 bp): (forward) 5′-GTG GGG CGC CCA GGC ACC A-3′ and (reverse) 5′-GCT CGG CCG TGG TGG TGA AGC-3′. The reaction was carried out for 30 cycles, using a 95 °C, 30-s denaturing step; a 57 °C, 30-s annealing step; and a 72 °C, 1-min extension step [18]. The IDVs were analyzed with computerized image analysis system (Fluor Chen 2.0). 2.5. Immunohistochemistry The spinal cord tissue of the rats in the sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group for 24 h groups post-injury was fixed by perfusion with 4% paraformaldehyde overnight at 4 °C, and then the tissues were precipitated by 20% and 30% sucrose, respectively. Following the instruction of SP-9000 HistostainTM-Plus Kit (Zhongshan Biotechnology, Beijing), the sections were blocked in reagent A for 20 min and then incubated with the rabbit polyclonal antibody anti-ZO-1 (diluted 1:50, Invitrogen, Life Technology) and antioccludin (diluted 1:50, Invitrogen, Life Technology) overnight at 4 °C. The sections were incubated with reagent B and C for 20 min at room temperature, respectively. At last, DAB was used to detect staining. For semi-quantitative measurements of ZO-1 and occludin expression, the slides were photographed and measured using a computer-aided image analyzing system. 2.6. Western blots analysis Western blots analysis was performed to investigate the protein expression of ZO-1, occludin, HO-1, TNF-α and NF-κB at 24 h post-SCI. Protein homogenates of spinal cord samples were prepared by rapid homogenization in lysis buffer (Beyotime Institute of Biotechnology, Beijing) for 2 h. Samples were centrifuged at 12,000g for 30 min at 4 °C. The protein concentration of soluble materials was measured by the BCA protein assay kit (Bicin-choninic Acid, Kangwei Biotechnology, Inc.). Protein lysates (40 μg per lane for each sample) were fractioned using the BoltTM Mini Gel Tank (4–12%). Then using iBlot Gel Transfer Device, we performed dry blotting of protein from mini gels with iBlot Gel Transfer Stacks for 7–9 min according to molecular weight of different protein. The membranes were blocked with blocking buffer (5% non-fat dairy milk dissolved in Tween–Tris-buffered saline, TTBS) for
2 h at room temperature. The blots were then incubated with rabbit polyclonal antibody anti-ZO-1 (dilution 1:100, Invitrogen, Life Technology), anti-occludin (dilution 1:100, Invitrogen, Life Technology), antiHO-1 (dilution 1:250, Santa Cruz), anti-TNF-α (dilution 1:200, Santa Cruz), anti-NF-κB (dilution 1:200, Santa Cruz) and β-actin (dilution 1:20000, Proteintech) antibody overnight at 4 °C. The ZO-1, occludin, HO-1 protein, TNF-α and NF-κB protein bands on these immunoblots were visualized using the enhanced chemiluminescene (ECL kit, Beyotime Institute of Biotechnology, Beijing). The ZO-1, occludin, HO1, TNF-α and NF-κB protein bands and β-actin bands were scanned using Chemi Imager software, and IDVs were calculated by Fluor Chen 2.0 software and normalized with that of β-actin.
2.7. Statistical analysis All data are presented as the mean ± SD. Statistical analysis of all protein and mRNA expression levels studies was performed using one-way analysis of variance (ANOVA) to compare the differences among the groups. For other measurements, the data were analyzed using paired Student's t test. All differences were considered significant statistically at P b 0.05.
3. Results 3.1. Effect of Cur on the permeability of BSCB There was no significant change in BSCB permeability between Cur treatment (75 mg/kg, i.p.) and SCI group (Fig. 1A). However, after treatment with Cur (150 or 300 mg/kg, i.p.), the content of Evans blue in spinal cord tissue was significantly decreased compared with SCI group at 12, 24 and 48 h after injury, respectively (Fig. 1B and C). In addition, pretreatment of ZnPP reduced the protective effect of Cur (150 or 300 mg/kg, i.p.) on BSCB at 24 and 48 h post-surgery (Fig. 1). Next, the dosage of Cur at 150 mg/kg was selected in the following experiment. Our results show that rats spontaneously recover the integrity of BSCB after incomplete SCI. We observed the effect of Cur (150 mg/kg) on EB leakages from BSCB at day 1, day 7 and day 21 post-SCI using immunofluorescence microscopy. It was observed that Cur could significantly inhibit the permeability of BSCB at day 1 and day 7 post-SCI, but there is no great difference in the permeability of BSCB between SCI group and SCI + Cur group at day 21 post-SCI (Fig. 1D and E).
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Fig. 2. Effect of Cur on motor function was assessed using the BBB scoring system every day up to the 21st day after SCI (n = 6, each). **P b 0.01 vs. SCI group. #P b 0.05 vs. SCI + Cur group.
D. Yu et al. / Journal of the Neurological Sciences 346 (2014) 51–59
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Fig. 4. Effects of Cur (150 mg/kg, i.p.) on TNF-α and NF-κB protein in spinal cord tissue at the edge of the resection site increased at 24 h after SCI. Lane 1: sham-operated group; lane 2: SCI group; lane 3: SCI + Cur group; lane 4: SCI + Cur + Znpp group. (A) Representative Western blots illustrating differences in 19-kDa band of TNF-α and the 65-kDa band of NF-κB. Relative integrated density value (IDV) analysis of TNF-α (B) and NF-κB protein (C) (n = 6, each). ⁎⁎P b 0.01 vs. sham-operated group. ##P b 0.01 vs. SCI group. *#P b 0.01 vs. SCI + Cur group.
0.2 0.1 0
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Fig. 3. Effects of Cur (150 mg/kg, i.p.) on TNF-α and NF-κB mRNA in the injured spinal cord at 24 h after SBI. Lane 1: sham-operated group; lane 2: SCI group; lane 3: SCI + Cur group; lane 4: SCI + Cur + Znpp group. Representative RT-PCR illustrating differences in the 413 bp band of TNF-α (A) and the 384 bp band of NF-κB mRNA (C). IDV analysis of TNF-α (B) and NF-κB mRNA (D) (n = 6, each). ⁎⁎P b 0.01, ⁎P b 0.05 vs. sham-operated group. ## P b 0.01 vs. SCI group. *#P b 0.01 vs. SCI + Cur group.
3.2. Effect of Cur on the motor function of rats The rats had significant dysfunction in hindlimb movement after SCI. Cur (150 mg/kg) markedly ameliorated the hindlimb motor dysfunction until the 21st day after SCI, suggesting that there is a long-term effect of Cur on SCI recovery. Moreover, the effect of Cur on the motor function could be greatly attenuated by the pretreatment of ZnPP (Fig. 2).
(150 mg/kg). The integrated density values (IDVs) of TNF-α mRNA with β-actin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group at 24 h after surgery were 0.21 ± 0.03, 0.75 ± 0.08, 0.47 ± 0.05 and 0.6 ± 0.05, respectively (Fig. 3B). The IDVs of NF-κB mRNA with β-actin were 0.25 ± 0.02, 0.82 ± 0.07, 0.45 ± 0.03 and 0.62 ± 0.05 (**P b 0.01,*P b 0.05 vs. sham-operated group; ##P b 0.01 vs. SCI group; ⁎#P b 0.01 vs. SCI + Cur group, Fig. 3D). The IDVs of TNF-α protein with β-actin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group at 24 h post-SCI were 0.23 ± 0.02, 0.62 ± 0.07, 0.35 ± 0.04 and 0.52 ± 0.06, respectively (Fig. 4B). The IDVs of NF-κB protein with β-actin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group at 24 h postSCI were 0.21 ± 0.02, 0.71 ± 0.08, 0.43 ± 0.04 and 0.59 ± 0.05, respectively (**P b 0.01 vs. sham-operated group; ##P b 0.01 vs. SCI group; ⁎#P b 0.05 vs. SCI + Cur group, Fig. 4C). 3.4. Effect of Cur on the expression level of tight junction protein
3.3. Effect of Cur on the expression of TNF-α and NF-κB mRNA and protein The expression of TNF-α and NF-κB mRNA and protein increased at 24 h after SCI, which was significantly attenuated by treatment with Cur
The up-regulation of TJ protein expression induced by Cur (150 mg/kg) was observed at 24 h after SCI (Fig. 5A). The IDV of the ZO-1 and occludin protein in SCI group was greatly lower than
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IDV of ZO-1 protein by Western blots
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3.5. Effect of Cur on the expression of HO-1 protein
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There were significant increases of protein levels of HO-1 at 24 h after SCI, which was further potentiated by Cur (150 mg/kg) treatment. However, induction of HO-1 protein expression was blocked by ZnPP (Fig.7A). The IDVs of HO-1 with β-actin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group at 24 h post-SCI were 0.21 ± 0.02, 0.64 ± 0.05, 0.85 ± 0.07 and 0.45 ± 0.04, respectively (**P b 0.01, *P b 0.05 vs. sham-operated group; ⁎#P b 0.01 vs. SCI + Cur group, Fig. 7B).
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Fig. 5. Effects of Cur on ZO-1 and occludin protein in the injured spinal cord site at 24 h after SCI. Lane 1: sham-operated group; lane 2: SCI group; lane 3: SCI + Cur group; lane 4: SCI + Cur + Znpp group. (A) Representative Western blots illustrating differences in ZO-1 and occludin. Relative integrated density value (IDV) analysis of ZO-1 (B) and occludin protein (C) (n = 6, each). ⁎⁎P b 0.01 vs. sham-operated group. ##P b 0.01, #P b 0.05 vs. SCI group. *#P b 0.01 vs. SCI + Cur group.
that in the sham-operated group at 24 h after SCI. The IDVs of ZO-1 with β-actin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group at 24 h after injury were 0.42 ± 0.04, 0.14 ± 0.02, 0.37 ± 0.03 and 0.25 ± 0.02 (Fig. 5B). The IDVs of occludin with β-actin were 0.45 ± 0.04, 0.22 ± 0.01, 0.48 ± 0.05 and 0.3 ± 0.02, respectively (**P b 0.01 vs. sham-operated group; ## P b 0.01, #P b 0.05 vs. SCI group; ⁎#P b 0.01 vs. SCI + Cur group, Fig. 5C). The roles of SCI-induced reduction of occludin and ZO-1 in the vascular of the gray matter were attenuated by Cur administration. However, ZnPP blocked the protective effects of Cur on SCI-induced decrease in occludin and ZO-1 expression in the vascular of gray matter (Fig. 6A–D). The mean optical density values of occludin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group were 0.22 ± 0.02, 0.14 ± 0.01, 0.2 ± 0.01 and 0.16 ± 0.01, respectively (Fig. 6E). The mean optical density values of ZO-1 in the abovementioned groups were 0.25 ± 0.02, 0.13 ± 0.01, 0.24 ± 0.02 and 0.17 ± 0.01, respectively (**P b 0.01, *P b 0.05 vs. sham-operated group; ##P b 0.01 vs. SCI group; ⁎#P b 0.01 vs. SCI + Cur group, Fig. 6F).
Cur at a dose of 150 or 300 mg/kg significantly reduced the increase in BSCB permeability at day 1 and day 2 after injury, whereas the other dose (75 mg/kg) did not significantly change it. The dose-dependent effect of Cur was in agreement with the findings of a recent study that has shown that Cur significantly reduced cerebral edema in subjects with surgical brain injury [8]. Therefore, we selected the dosage of 150 mg/kg in the present experiment. Spinal cord homeostasis is the basis for maintaining the normal function of neurons in spinal cord tissue. The BSCB, which is a metabolism barrier between the spinal cord tissue and blood circulation, strictly controls the spinal microenvironment. Early repair of the BSCB has important significance for SCI treatment. However, previous studies have focused more on the mechanism of nerve cell apoptosis after SCI and have ignored the idea that improvements in BSCB function are key to recovery after SCI. Some studies have suggested the effects of Cur on acute spinal cord recovery (up to 7 days, see [9]). However, no report has analyzed the effects of Cur on long-term recovery. It will be of great interest to know whether Cur has any long-term effects on SCI recovery. In rats with SCI, gross motor recovery occurs in the first 2 weeks after SCI and plateaus by 3 weeks post-SCI [19]. In the present study, motor recovery of rats with SCI was assessed with the Basso, Beattie, and Bresnahan scoring system. Cur (150 mg/kg) significantly ameliorated the hind limb motor disturbances up to 21 days after SCI, and pretreatment with zinc protoporphyrin reduced the effect of Cur on motor function, suggesting that Cur exerts long-term effects on SCI recovery via HO-1 induction. At the same time, we investigated the time course of effects of Cur on BSCB permeability. Cur significantly inhibited the BSCB permeability at day 1 and day 7 after SCI, but there was no significant difference in BSCB permeability between the SCI and SCI + Cur groups at day 21 after SCI; this suggests that early repair of the BSCB has an important effect on the secondary nerve injury induced by SCI. On the basis of our results, we concluded that the Cur-induced early repair of the BSCB might be helpful for motor recovery. The protective effects of Cur on SCI-induced BSCB disruption still need to be investigated. TNF-α participates in glia-mediated inflammation through activation of the NF-κB signaling pathway [20]. TNF-α, an inflammatory cytokine, enhances permeability of the BBB/BSCB. TNF-α alters the TJ structure and protein distribution in monoculture endothelial models of BBB/BSCB. Increases in BBB/BSCB permeability following TNF-α treatment are related to activation of the NF-κB-mediated signaling pathway in the cerebral microvasculature [21]. TNF-α can decrease the expression of TJ proteins by increasing the p65 subunit of NF-κB [22]. Previous results have suggested that NF-κB is a major signal transducer via which TNF-α affects BBB/BSCB permeability. In the present study, TNF-α and NF-κB expression was markedly increased at 24 h after SCI, and this increase was significantly attenuated after Cur treatment. These results suggest that Cur influences BSCB permeability by downregulating the expression of TNF-α and NF-κB at the boundary of the resection site after SCI. The BSCB is mainly composed of spinal cord vascular endothelial cells, basement membrane, and astrocyte foot processes as well as cerebral microvascular endothelial cells, lack of window and pinocytotic vesicles [23]. After SCI, TJ damage and number of pinocytotic vesicles
D. Yu et al. / Journal of the Neurological Sciences 346 (2014) 51–59
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F ## ## * *#
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Fig. 6. Effects of Cur on ZO-1 and occludin protein in the gray matter of spinal cord at 24 h after SCI by immunohistochemical method. A: sham-operated group; B: SCI group; C: SCI + Cur group; D: SCI + Cur + Znpp group. Mean optical density value (IDV) analysis of occludin (E) and ZO-1 protein (F) (n = 6, each). ⁎⁎P b 0.01 vs. sham-operated group. ##P b 0.01 vs. SCI group. *#P b 0.01 vs. SCI + Cur group. Scale bar: 20 μm.
in the BSCB increase, resulting in disruption of BSCB permeability [24]. The BSCB displays a unique phenotype characterized by the presence of TJs [23,25]. A TJ is an intercellular adhesion complex that forms close contact between adjacent cells. TJs are composed of complicated TJ proteins, including transmembrane proteins, members of the peripheral membrane protein family, and adhesion molecules [26]. Dysfunction of TJs, which represent BSCB disruption, is most frequently seen during SCI [27]. To study the effects of Cur on BSCB function, we examined the effects of Cur on TJ proteins in the injured spinal cord. We observed that Cur upregulated the expression of TJ protein in the injured spinal cord. This suggested a role for Cur in protection against BSCB injury in SCI, and this corresponded with other models of brain injury. Together, these analyses provide compelling evidence that Cur could directly promote and enhance the integrity of BSCB during SCI independent of its anti-inflammatory effects. However, the mechanism underlying the enhancement of TJ protein expression after Cur treatment is not fully understood. HO-1 is involved in the rate-limiting step in the oxidative degradation of heme. HO-1 expression is upregulated in response to oxidative stress, and it catalyzes the degradation of pro-oxidant heme to carbon monoxide, iron, and bilirubin [28]. HO-1 expression is an adaptive and protective response to oxidative stress. HO-1 can be induced by a variety of stimuli and various agents that cause oxidative stress, and it protects cells from the oxidative damage caused by reactive oxygen species (ROS) [29]. Thus, the HO-1 pathway is of crucial for preservation of
BBB/BSCB integrity in oxidative stress, and it acts in synchrony with other enzymatic systems for maintaining cellular homeostasis [28,30]. In addition, a study reported that transfection of a mutant HO-1 protein without enzymatic activity significantly attenuated oxidative stressmediated cellular injury [31], suggesting that the protein molecule may also have an important role in the regulation of intracellular signal transduction independent of its enzymatic activity. Therefore, we examined the effects of Cur on HO-1 protein expression. Cur is a naturally occurring compound that is known to induce HO-1 production, although the underlying mechanism has not been fully understood. Recently, Cur has been shown to exert cytoprotective properties by inducing the protective protein HO-1 [32–34]. Wang et al. demonstrated that HO-1 immunoreactivity is primarily observed in vascular-like structures [35]. Bone marrow stem cells overexpressing HO-1 not only inhibit the damage induced by high glucose concentration but also promote proliferation and migration of vascular endothelial cells [36]. HO-1 is cytoprotective during oxidative stress [25,33], and HO-1 induction is a generalized response to oxidative stress [29]. ROS can timedependently regulate the expression and distribution of TJ protein and then induce an increase in BBB permeability through the ROS/ RhoA/phosphatidylinositol 3-kinase/protein kinase B signaling pathway [37]. Previous studies have shown that Cur prevents middle cerebral artery occlusion-induced oxidative damage [7] and protects human intestinal epithelial cells against the ROS-induced disruption of TJ and the barrier by inducing HO-1 expression [10]. HO-1
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A
University, No. XZJJ20130241; National Natural Science Foundation of China, Nos. 81171799 and 81272074; and National Natural Science Foundation of Liaoning Province, No. 201102121.
References
IDV of HO-1 protein by western blots
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Fig. 7. Effects of Cur on HO-1 protein in the injured spinal cord site at 24 h after SCI. Lane 1: sham-operated group; lane 2: SCI group; lane 3: SCI + Cur group; lane 4: SCI + Cur + Znpp group. (A) Representative Western blots illustrating differences in HO-1. (B) Relative integrated density value (IDV) analysis of HO-1 protein (n = 6, each). ⁎⁎P b 0.01 vs. sham-operated group. #P b 0.05 vs. SCI group. *#P b 0.01 vs. SCI + Cur group.
functions as a suppressor of TNF-α signaling, not only by inhibiting the expression of adhesion molecules and generation of interleukin-6 but also by diminishing intracellular ROS production and NF-κB activation in both cultured human tracheal smooth muscle cells and the airways of mice [38]. We hypothesize that Cur could increase TJ protein expression via the HO-1/ROS signaling pathway. In this study, we demonstrated that Cur induced HO-1 expression and restored occludin and ZO-1 protein levels that were induced by SCI. Zinc protoporphyrin, which is a commonly used specific HO-1 inhibitor, reversed the protective role of Cur, suggesting that Cur has a protective effect that, at least in part, depends on an antioxidant mechanism based on HO-1 induction. In conclusion, our findings indicated that Cur effectively and doseindependently protected the BSCB through attenuation of inflammatory factors and TJ protein expression in the injured spinal cord. In addition, the effects of Cur on motor function after SCI were observed over the long term. However, Cur has been shown to inhibit the extrinsic and intrinsic pathways of blood coagulation and increase the chance of bleeding by inhibiting factor Xa and thrombin generation in human umbilical vein endothelial cells [39]. Therefore, the time and dosage of Cur should be carefully considered for each patient with SCI. More studies are essential to consider the possible therapeutic effects of Cur in these patients. There now appears to be new and potentially powerful opportunities for treating acute SCI by targeting the vascular responses. Conflict of interest We state that there is no conflict of interest. Acknowledgments This work was supported by Doctoral Scientific Research Starting Foundation of Liaoning Province, No. 20121096; Scientific Research Starting Foundation for PH.D. and Returned Overseas Teacher of Liaoning Medical University, No. Y2012B011; Program for Liaoning Excellent Talents in University, No. LR2013091; Special Funds for Clinical Medicine Construction of the Principal's Fund of the Liaoning Medical
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Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Curcumin improves the integrity of blood–spinal cord barrier after compressive spinal cord injury in rats De-shui Yu a, Yang Cao a,⁎, Xi-fan Mei a, Yan-feng Wang b, Zhong-kai Fan a, Yan-song Wang a, Gang Lv a a b
Department of Orthopaedics, The First Affiliated Hospital, Liaoning Medical University, People Street No. 5-2, GuTa District, Jinzhou 121001, Liaoning Province, People's Republic of China Department of Orthopaedics, the First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, People's Republic of China
a r t i c l e
i n f o
Article history: Received 13 October 2013 Received in revised form 23 July 2014 Accepted 25 July 2014 Available online 4 August 2014 Keywords: Curcumin Spinal cord injury Blood–spinal cord barrier Tight junction Heme oxygenase-1 Tight junction protein
a b s t r a c t Previous studies have shown that curcumin (Cur) can produce potent neuroprotective effects against damage due to spinal cord injury (SCI). However, whether Cur can preserve the function of the blood–spinal cord barrier (BSCB) is unclear. The present study was performed to investigate the mechanism underlying BSCB permeability changes, which were induced by treatment with Cur (75, 150, and 300 mg/kg, i.p.) after compressive SCI in rats. BSCB permeability was evaluated by Evans blue leakage. Motor recovery of rats with SCI was assessed using the Basso, Beattie, and Bresnahan scoring system every day until the 21st days post-injury. The protein levels of heme oxygenase-1 (HO-1), tight junction protein, and inflammatory factors were analyzed by western blots. The expression of the inflammatory factors tumor necrosis factor-α (TNF-α) and nuclear factor-kappaB (NF-κB) mRNA was determined with reverse transcription-polymerase chain reactions. Treatment with Cur (150 and 300 mg/kg) significantly reduced Evans blue leakage into the spinal cord tissue at 24 h after SCI. Cur (150 mg/kg) significantly increased HO-1 protein expression. The levels of TNF-α and NF-κB mRNA and protein greatly increased at 24 h after SCI, and this increase was significantly attenuated by Cur treatment. ZO-1 and occludin expression was upregulated by Cur (150 mg/kg) treatment after SCI, and this effect was blocked by the HO-1 inhibitor zinc protoporphyrin. Long-term effects of Cur on motor recovery after SCI were observed. Our results indicated that Cur can improve motor function after SCI, which could correlate with improvements in BSCB integrity. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Spinal cord injury (SCI) is one of the most devastating traumatic conditions that induce primary and secondary tissue damage, resulting in postoperative complications, including spinal cord edema and cell death in the injured areas [1]. Recently, it has been reported that blood–spinal cord barrier (BSCB) disruption is associated with increased mortality after endovascular therapy, and improvements in the BSCB functioning can significantly reduce secondary nerve injury [2,3], suggesting that early repairs of the BSCB have important significance in the treatment of SCI. Therefore, drugs that reduce BSCB breakdown might improve outcomes of patients with SCI by limiting more aggressive surgical resections in confined spaces. Curcumin (Cur), which is a major active component of the food ingredient turmeric, is isolated from the powdered dry rhizome of Curcuma longa Linn. This component has a variety of pharmacological activities, including anti-oxidative, anti-inflammatory, anti-carcinogenic, and anti-HIV effects [4–6]. Previous studies have revealed that Cur exerts
⁎ Corresponding author. Tel./fax: +86 4164197717. E-mail address: [email protected] (Y. Cao).
http://dx.doi.org/10.1016/j.jns.2014.07.056 0022-510X/© 2014 Elsevier B.V. All rights reserved.
a neuroprotective role in diseases of the central nervous system, such as cerebral ischemia and traumatic brain injury [7,8]. Cur treatment has been shown to reduce edema and inflammation in the lesion areas of rats with SCI, suggesting its potential as a therapeutic agent [9]. However, the effects of Cur on the BSCB are still unclear. Recently, numerous studies have shown that the induction of heme oxygenase-1 (HO-1) is an important cellular protective mechanism against oxidative injury [10]. Due to the cumulative effects of HO-1 on heme catabolism and the generation of biologically active downstream products, HO-1 induction might serve as a protective mechanism against inflammatory injury. Inflammatory factors have been shown to increase BSCB permeability by timedependently modulating the expression and distribution of tight junction (TJ) proteins [11,12]. Cur, which is a naturally occurring potent inducer of HO-1 [13,14], provides protection against the release of inflammatory factors [15]. Recently, Cur has been demonstrated to protect human intestinal epithelial cells against the H2O2-induced disruption of TJ and barrier dysfunction through the HO-1 pathway [10]. Recently, Wang et al. have demonstrated that Cur preserves the permeability of the blood–brain barrier (BBB) during hypoxia by upregulating HO-1 expression in brain microvascular endothelial cells [16]. Therefore, we hypothesized that Cur may limit the SCI-induced disruption of TJs and barrier dysfunction through the HO-1 pathway. To test this hypothesis, we utilized a
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compressive SCI model in rat and evaluated the effects of Cur on HO-1, inflammatory factors, TJ disruption, BSCB dysfunction, and motor function that were induced by SCI. 2. Materials and methods 2.1. Establishment of spinal cord compression injury model in rats The adult male Sprang–Dawley rats weighing 210–260 g were purchased from the Experimental Animals Center of Liaoning Medical University. All animal experiments were conducted in accordance with the National Institute of Health Guide on the Care and Use of Laboratory Animals. The spinal cords were injured by arterial clamp with a closing force of 20 g (5 min) at thoracic level 8–10 (T8–10), using an established compression model. Rats were randomly and blindly divided into four groups, including sham-operated group, SCI group, SCI + Cur group and SCI + Cur +
ZnPP group. Sham-operated group, sham surgery included general anesthesia, skin incision, and laminectomy but clip compression was not applied; SCI group, rats were given a compression injury as the abovementioned method; SCI + Cur group, rats received immediately the treatment of Cur (Sigma-Aldrich) at the doses of 75, 150 and 300 mg/kg (i.p.) at 20 min after SCI and were given the administration of Cur every 24 h until the 3rd day after SCI, respectively; SCI + Cur + ZnPP group, rats were injected 25 μmol/kg of Znpp (i.p.) at 2 h before SCI and then were given the treatment of Cur (150 mg/kg, i.p.) every 24 h until the 3rd day after SCI. Six rats of each group were used in the following experiments respectively. 2.2. Measurement of BSCB permeability by Evans blue leakage BSCB permeability was quantitatively evaluated by extravasation of Evans blue (EB) as a marker of albumin extravasation. Briefly, rats for different time point group were injected 2% EB (2 ml/kg, iv). Two
A
12 hr
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48 hr
B #
# **
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**
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48 hr
C #
# **
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Fig. 1. The permeability of blood–spinal cord barrier was assessed by Evans blue extravasation (μg/g spinal cord tissue) and immunofluorescence at different time point in SCI group, SCI + Cur group (150 mg/kg, i.p.) and SCI + Cur + Znpp group. Data present means ± S.D. (n = 6, each). ⁎⁎P b 0.01 vs. SCI group. #P b 0.05 vs. SCI + Cur group.
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D # #
** *
E
1 day
7 day
21 day
# **
Fig. 1 (continued).
hours after EB injection, rats in different groups were transcardially perfused with 0.9% normal saline until colorless perfusion fluid was obtained from the right atrium. The injured spinal cord tissues were weighed and immersed in formamide (1 ml/100 mg) at 60 °C for 24 h. The optical density of the supernatant was examined with enzyme-labelled meter (at 620 nm, TECAN). The quantitative calculation of the dye content in the spinal cord tissue was based on the external standards dissolved in the same solvent. Rats for 1, 7 and 21 day post-SCI group were fixed by perfusion with 4% paraformaldehyde at 2 h after EB injection. The spinal cord tissues in different group were cut into 12 μm thickness at −27 °C using frozensection machine (Thermo Shandon). Evans blue leakage at the vascular level was also observed at day 1, day 7 and day 21 groups using immunofluorescence microscopy (IX71, Olympus).
2.3. Grading of motor disturbance Evaluation of motor dysfunction was graded using the modified murine Basso, Beattie, and Bresnahan (BBB) scoring system [17]. Motor function of rats that suffered from spinal cord compression injury was evaluated every day until the 21st day after SCI. 2.4. RT-PCR analysis The mRNA expression levels of tumor necrosis factor-α (TNF-α) and nuclear factor-kappaB (NF-κB) were determined by RT-PCR for 24 h groups post-SCI in rats. Total RNA was isolated from the injured spinal cord samples and purified using RNAout kit (Takara). Following the manufacturer's instructions, cDNAs were obtained from 1 μg of total
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RNA from each example. The oligonucleotide primers were as follows: NF-κB (P65) (381 bp): (forward) 5′CCT ATC CAC GAC AAC CTT GC 3′ and (reverse) 5′CAT AGA TGC TGC TGA CCC AAC 3′; TNF-α (413 bp): (forward) 5′CCC CAA GTG ACA AGC CAG TA 3′ and (reverse) 5′CAA AGT CCA GAT TAG GCA GAT 3′; β-actin (493 bp): (forward) 5′-GTG GGG CGC CCA GGC ACC A-3′ and (reverse) 5′-GCT CGG CCG TGG TGG TGA AGC-3′. The reaction was carried out for 30 cycles, using a 95 °C, 30-s denaturing step; a 57 °C, 30-s annealing step; and a 72 °C, 1-min extension step [18]. The IDVs were analyzed with computerized image analysis system (Fluor Chen 2.0). 2.5. Immunohistochemistry The spinal cord tissue of the rats in the sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group for 24 h groups post-injury was fixed by perfusion with 4% paraformaldehyde overnight at 4 °C, and then the tissues were precipitated by 20% and 30% sucrose, respectively. Following the instruction of SP-9000 HistostainTM-Plus Kit (Zhongshan Biotechnology, Beijing), the sections were blocked in reagent A for 20 min and then incubated with the rabbit polyclonal antibody anti-ZO-1 (diluted 1:50, Invitrogen, Life Technology) and antioccludin (diluted 1:50, Invitrogen, Life Technology) overnight at 4 °C. The sections were incubated with reagent B and C for 20 min at room temperature, respectively. At last, DAB was used to detect staining. For semi-quantitative measurements of ZO-1 and occludin expression, the slides were photographed and measured using a computer-aided image analyzing system. 2.6. Western blots analysis Western blots analysis was performed to investigate the protein expression of ZO-1, occludin, HO-1, TNF-α and NF-κB at 24 h post-SCI. Protein homogenates of spinal cord samples were prepared by rapid homogenization in lysis buffer (Beyotime Institute of Biotechnology, Beijing) for 2 h. Samples were centrifuged at 12,000g for 30 min at 4 °C. The protein concentration of soluble materials was measured by the BCA protein assay kit (Bicin-choninic Acid, Kangwei Biotechnology, Inc.). Protein lysates (40 μg per lane for each sample) were fractioned using the BoltTM Mini Gel Tank (4–12%). Then using iBlot Gel Transfer Device, we performed dry blotting of protein from mini gels with iBlot Gel Transfer Stacks for 7–9 min according to molecular weight of different protein. The membranes were blocked with blocking buffer (5% non-fat dairy milk dissolved in Tween–Tris-buffered saline, TTBS) for
2 h at room temperature. The blots were then incubated with rabbit polyclonal antibody anti-ZO-1 (dilution 1:100, Invitrogen, Life Technology), anti-occludin (dilution 1:100, Invitrogen, Life Technology), antiHO-1 (dilution 1:250, Santa Cruz), anti-TNF-α (dilution 1:200, Santa Cruz), anti-NF-κB (dilution 1:200, Santa Cruz) and β-actin (dilution 1:20000, Proteintech) antibody overnight at 4 °C. The ZO-1, occludin, HO-1 protein, TNF-α and NF-κB protein bands on these immunoblots were visualized using the enhanced chemiluminescene (ECL kit, Beyotime Institute of Biotechnology, Beijing). The ZO-1, occludin, HO1, TNF-α and NF-κB protein bands and β-actin bands were scanned using Chemi Imager software, and IDVs were calculated by Fluor Chen 2.0 software and normalized with that of β-actin.
2.7. Statistical analysis All data are presented as the mean ± SD. Statistical analysis of all protein and mRNA expression levels studies was performed using one-way analysis of variance (ANOVA) to compare the differences among the groups. For other measurements, the data were analyzed using paired Student's t test. All differences were considered significant statistically at P b 0.05.
3. Results 3.1. Effect of Cur on the permeability of BSCB There was no significant change in BSCB permeability between Cur treatment (75 mg/kg, i.p.) and SCI group (Fig. 1A). However, after treatment with Cur (150 or 300 mg/kg, i.p.), the content of Evans blue in spinal cord tissue was significantly decreased compared with SCI group at 12, 24 and 48 h after injury, respectively (Fig. 1B and C). In addition, pretreatment of ZnPP reduced the protective effect of Cur (150 or 300 mg/kg, i.p.) on BSCB at 24 and 48 h post-surgery (Fig. 1). Next, the dosage of Cur at 150 mg/kg was selected in the following experiment. Our results show that rats spontaneously recover the integrity of BSCB after incomplete SCI. We observed the effect of Cur (150 mg/kg) on EB leakages from BSCB at day 1, day 7 and day 21 post-SCI using immunofluorescence microscopy. It was observed that Cur could significantly inhibit the permeability of BSCB at day 1 and day 7 post-SCI, but there is no great difference in the permeability of BSCB between SCI group and SCI + Cur group at day 21 post-SCI (Fig. 1D and E).
**
**
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** #
** #
** #
** #
**
#
#
Fig. 2. Effect of Cur on motor function was assessed using the BBB scoring system every day up to the 21st day after SCI (n = 6, each). **P b 0.01 vs. SCI group. #P b 0.05 vs. SCI + Cur group.
D. Yu et al. / Journal of the Neurological Sciences 346 (2014) 51–59
A
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B B 0.9 IDV of TNF-α mRNA by RT-PCR
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Fig. 4. Effects of Cur (150 mg/kg, i.p.) on TNF-α and NF-κB protein in spinal cord tissue at the edge of the resection site increased at 24 h after SCI. Lane 1: sham-operated group; lane 2: SCI group; lane 3: SCI + Cur group; lane 4: SCI + Cur + Znpp group. (A) Representative Western blots illustrating differences in 19-kDa band of TNF-α and the 65-kDa band of NF-κB. Relative integrated density value (IDV) analysis of TNF-α (B) and NF-κB protein (C) (n = 6, each). ⁎⁎P b 0.01 vs. sham-operated group. ##P b 0.01 vs. SCI group. *#P b 0.01 vs. SCI + Cur group.
0.2 0.1 0
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Fig. 3. Effects of Cur (150 mg/kg, i.p.) on TNF-α and NF-κB mRNA in the injured spinal cord at 24 h after SBI. Lane 1: sham-operated group; lane 2: SCI group; lane 3: SCI + Cur group; lane 4: SCI + Cur + Znpp group. Representative RT-PCR illustrating differences in the 413 bp band of TNF-α (A) and the 384 bp band of NF-κB mRNA (C). IDV analysis of TNF-α (B) and NF-κB mRNA (D) (n = 6, each). ⁎⁎P b 0.01, ⁎P b 0.05 vs. sham-operated group. ## P b 0.01 vs. SCI group. *#P b 0.01 vs. SCI + Cur group.
3.2. Effect of Cur on the motor function of rats The rats had significant dysfunction in hindlimb movement after SCI. Cur (150 mg/kg) markedly ameliorated the hindlimb motor dysfunction until the 21st day after SCI, suggesting that there is a long-term effect of Cur on SCI recovery. Moreover, the effect of Cur on the motor function could be greatly attenuated by the pretreatment of ZnPP (Fig. 2).
(150 mg/kg). The integrated density values (IDVs) of TNF-α mRNA with β-actin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group at 24 h after surgery were 0.21 ± 0.03, 0.75 ± 0.08, 0.47 ± 0.05 and 0.6 ± 0.05, respectively (Fig. 3B). The IDVs of NF-κB mRNA with β-actin were 0.25 ± 0.02, 0.82 ± 0.07, 0.45 ± 0.03 and 0.62 ± 0.05 (**P b 0.01,*P b 0.05 vs. sham-operated group; ##P b 0.01 vs. SCI group; ⁎#P b 0.01 vs. SCI + Cur group, Fig. 3D). The IDVs of TNF-α protein with β-actin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group at 24 h post-SCI were 0.23 ± 0.02, 0.62 ± 0.07, 0.35 ± 0.04 and 0.52 ± 0.06, respectively (Fig. 4B). The IDVs of NF-κB protein with β-actin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group at 24 h postSCI were 0.21 ± 0.02, 0.71 ± 0.08, 0.43 ± 0.04 and 0.59 ± 0.05, respectively (**P b 0.01 vs. sham-operated group; ##P b 0.01 vs. SCI group; ⁎#P b 0.05 vs. SCI + Cur group, Fig. 4C). 3.4. Effect of Cur on the expression level of tight junction protein
3.3. Effect of Cur on the expression of TNF-α and NF-κB mRNA and protein The expression of TNF-α and NF-κB mRNA and protein increased at 24 h after SCI, which was significantly attenuated by treatment with Cur
The up-regulation of TJ protein expression induced by Cur (150 mg/kg) was observed at 24 h after SCI (Fig. 5A). The IDV of the ZO-1 and occludin protein in SCI group was greatly lower than
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IDV of ZO-1 protein by Western blots
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3.5. Effect of Cur on the expression of HO-1 protein
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IDV of Occludin protein by Western blots
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4. Discussion
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There were significant increases of protein levels of HO-1 at 24 h after SCI, which was further potentiated by Cur (150 mg/kg) treatment. However, induction of HO-1 protein expression was blocked by ZnPP (Fig.7A). The IDVs of HO-1 with β-actin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group at 24 h post-SCI were 0.21 ± 0.02, 0.64 ± 0.05, 0.85 ± 0.07 and 0.45 ± 0.04, respectively (**P b 0.01, *P b 0.05 vs. sham-operated group; ⁎#P b 0.01 vs. SCI + Cur group, Fig. 7B).
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SCI+Cur+ZnPP
Fig. 5. Effects of Cur on ZO-1 and occludin protein in the injured spinal cord site at 24 h after SCI. Lane 1: sham-operated group; lane 2: SCI group; lane 3: SCI + Cur group; lane 4: SCI + Cur + Znpp group. (A) Representative Western blots illustrating differences in ZO-1 and occludin. Relative integrated density value (IDV) analysis of ZO-1 (B) and occludin protein (C) (n = 6, each). ⁎⁎P b 0.01 vs. sham-operated group. ##P b 0.01, #P b 0.05 vs. SCI group. *#P b 0.01 vs. SCI + Cur group.
that in the sham-operated group at 24 h after SCI. The IDVs of ZO-1 with β-actin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group at 24 h after injury were 0.42 ± 0.04, 0.14 ± 0.02, 0.37 ± 0.03 and 0.25 ± 0.02 (Fig. 5B). The IDVs of occludin with β-actin were 0.45 ± 0.04, 0.22 ± 0.01, 0.48 ± 0.05 and 0.3 ± 0.02, respectively (**P b 0.01 vs. sham-operated group; ## P b 0.01, #P b 0.05 vs. SCI group; ⁎#P b 0.01 vs. SCI + Cur group, Fig. 5C). The roles of SCI-induced reduction of occludin and ZO-1 in the vascular of the gray matter were attenuated by Cur administration. However, ZnPP blocked the protective effects of Cur on SCI-induced decrease in occludin and ZO-1 expression in the vascular of gray matter (Fig. 6A–D). The mean optical density values of occludin in sham-operated group, SCI group, SCI + Cur group and SCI + Cur + ZnPP group were 0.22 ± 0.02, 0.14 ± 0.01, 0.2 ± 0.01 and 0.16 ± 0.01, respectively (Fig. 6E). The mean optical density values of ZO-1 in the abovementioned groups were 0.25 ± 0.02, 0.13 ± 0.01, 0.24 ± 0.02 and 0.17 ± 0.01, respectively (**P b 0.01, *P b 0.05 vs. sham-operated group; ##P b 0.01 vs. SCI group; ⁎#P b 0.01 vs. SCI + Cur group, Fig. 6F).
Cur at a dose of 150 or 300 mg/kg significantly reduced the increase in BSCB permeability at day 1 and day 2 after injury, whereas the other dose (75 mg/kg) did not significantly change it. The dose-dependent effect of Cur was in agreement with the findings of a recent study that has shown that Cur significantly reduced cerebral edema in subjects with surgical brain injury [8]. Therefore, we selected the dosage of 150 mg/kg in the present experiment. Spinal cord homeostasis is the basis for maintaining the normal function of neurons in spinal cord tissue. The BSCB, which is a metabolism barrier between the spinal cord tissue and blood circulation, strictly controls the spinal microenvironment. Early repair of the BSCB has important significance for SCI treatment. However, previous studies have focused more on the mechanism of nerve cell apoptosis after SCI and have ignored the idea that improvements in BSCB function are key to recovery after SCI. Some studies have suggested the effects of Cur on acute spinal cord recovery (up to 7 days, see [9]). However, no report has analyzed the effects of Cur on long-term recovery. It will be of great interest to know whether Cur has any long-term effects on SCI recovery. In rats with SCI, gross motor recovery occurs in the first 2 weeks after SCI and plateaus by 3 weeks post-SCI [19]. In the present study, motor recovery of rats with SCI was assessed with the Basso, Beattie, and Bresnahan scoring system. Cur (150 mg/kg) significantly ameliorated the hind limb motor disturbances up to 21 days after SCI, and pretreatment with zinc protoporphyrin reduced the effect of Cur on motor function, suggesting that Cur exerts long-term effects on SCI recovery via HO-1 induction. At the same time, we investigated the time course of effects of Cur on BSCB permeability. Cur significantly inhibited the BSCB permeability at day 1 and day 7 after SCI, but there was no significant difference in BSCB permeability between the SCI and SCI + Cur groups at day 21 after SCI; this suggests that early repair of the BSCB has an important effect on the secondary nerve injury induced by SCI. On the basis of our results, we concluded that the Cur-induced early repair of the BSCB might be helpful for motor recovery. The protective effects of Cur on SCI-induced BSCB disruption still need to be investigated. TNF-α participates in glia-mediated inflammation through activation of the NF-κB signaling pathway [20]. TNF-α, an inflammatory cytokine, enhances permeability of the BBB/BSCB. TNF-α alters the TJ structure and protein distribution in monoculture endothelial models of BBB/BSCB. Increases in BBB/BSCB permeability following TNF-α treatment are related to activation of the NF-κB-mediated signaling pathway in the cerebral microvasculature [21]. TNF-α can decrease the expression of TJ proteins by increasing the p65 subunit of NF-κB [22]. Previous results have suggested that NF-κB is a major signal transducer via which TNF-α affects BBB/BSCB permeability. In the present study, TNF-α and NF-κB expression was markedly increased at 24 h after SCI, and this increase was significantly attenuated after Cur treatment. These results suggest that Cur influences BSCB permeability by downregulating the expression of TNF-α and NF-κB at the boundary of the resection site after SCI. The BSCB is mainly composed of spinal cord vascular endothelial cells, basement membrane, and astrocyte foot processes as well as cerebral microvascular endothelial cells, lack of window and pinocytotic vesicles [23]. After SCI, TJ damage and number of pinocytotic vesicles
D. Yu et al. / Journal of the Neurological Sciences 346 (2014) 51–59
E
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F ## ## * *#
* *#
** **
Fig. 6. Effects of Cur on ZO-1 and occludin protein in the gray matter of spinal cord at 24 h after SCI by immunohistochemical method. A: sham-operated group; B: SCI group; C: SCI + Cur group; D: SCI + Cur + Znpp group. Mean optical density value (IDV) analysis of occludin (E) and ZO-1 protein (F) (n = 6, each). ⁎⁎P b 0.01 vs. sham-operated group. ##P b 0.01 vs. SCI group. *#P b 0.01 vs. SCI + Cur group. Scale bar: 20 μm.
in the BSCB increase, resulting in disruption of BSCB permeability [24]. The BSCB displays a unique phenotype characterized by the presence of TJs [23,25]. A TJ is an intercellular adhesion complex that forms close contact between adjacent cells. TJs are composed of complicated TJ proteins, including transmembrane proteins, members of the peripheral membrane protein family, and adhesion molecules [26]. Dysfunction of TJs, which represent BSCB disruption, is most frequently seen during SCI [27]. To study the effects of Cur on BSCB function, we examined the effects of Cur on TJ proteins in the injured spinal cord. We observed that Cur upregulated the expression of TJ protein in the injured spinal cord. This suggested a role for Cur in protection against BSCB injury in SCI, and this corresponded with other models of brain injury. Together, these analyses provide compelling evidence that Cur could directly promote and enhance the integrity of BSCB during SCI independent of its anti-inflammatory effects. However, the mechanism underlying the enhancement of TJ protein expression after Cur treatment is not fully understood. HO-1 is involved in the rate-limiting step in the oxidative degradation of heme. HO-1 expression is upregulated in response to oxidative stress, and it catalyzes the degradation of pro-oxidant heme to carbon monoxide, iron, and bilirubin [28]. HO-1 expression is an adaptive and protective response to oxidative stress. HO-1 can be induced by a variety of stimuli and various agents that cause oxidative stress, and it protects cells from the oxidative damage caused by reactive oxygen species (ROS) [29]. Thus, the HO-1 pathway is of crucial for preservation of
BBB/BSCB integrity in oxidative stress, and it acts in synchrony with other enzymatic systems for maintaining cellular homeostasis [28,30]. In addition, a study reported that transfection of a mutant HO-1 protein without enzymatic activity significantly attenuated oxidative stressmediated cellular injury [31], suggesting that the protein molecule may also have an important role in the regulation of intracellular signal transduction independent of its enzymatic activity. Therefore, we examined the effects of Cur on HO-1 protein expression. Cur is a naturally occurring compound that is known to induce HO-1 production, although the underlying mechanism has not been fully understood. Recently, Cur has been shown to exert cytoprotective properties by inducing the protective protein HO-1 [32–34]. Wang et al. demonstrated that HO-1 immunoreactivity is primarily observed in vascular-like structures [35]. Bone marrow stem cells overexpressing HO-1 not only inhibit the damage induced by high glucose concentration but also promote proliferation and migration of vascular endothelial cells [36]. HO-1 is cytoprotective during oxidative stress [25,33], and HO-1 induction is a generalized response to oxidative stress [29]. ROS can timedependently regulate the expression and distribution of TJ protein and then induce an increase in BBB permeability through the ROS/ RhoA/phosphatidylinositol 3-kinase/protein kinase B signaling pathway [37]. Previous studies have shown that Cur prevents middle cerebral artery occlusion-induced oxidative damage [7] and protects human intestinal epithelial cells against the ROS-induced disruption of TJ and the barrier by inducing HO-1 expression [10]. HO-1
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A
University, No. XZJJ20130241; National Natural Science Foundation of China, Nos. 81171799 and 81272074; and National Natural Science Foundation of Liaoning Province, No. 201102121.
References
IDV of HO-1 protein by western blots
B
1
** #
0.9 0.8
**
0.7 0.6 0.5
*#
0.4 0.3 0.2 0.1 0
Sham
SCI
SCI+Cur
SCI+Cur+ZnPP
Fig. 7. Effects of Cur on HO-1 protein in the injured spinal cord site at 24 h after SCI. Lane 1: sham-operated group; lane 2: SCI group; lane 3: SCI + Cur group; lane 4: SCI + Cur + Znpp group. (A) Representative Western blots illustrating differences in HO-1. (B) Relative integrated density value (IDV) analysis of HO-1 protein (n = 6, each). ⁎⁎P b 0.01 vs. sham-operated group. #P b 0.05 vs. SCI group. *#P b 0.01 vs. SCI + Cur group.
functions as a suppressor of TNF-α signaling, not only by inhibiting the expression of adhesion molecules and generation of interleukin-6 but also by diminishing intracellular ROS production and NF-κB activation in both cultured human tracheal smooth muscle cells and the airways of mice [38]. We hypothesize that Cur could increase TJ protein expression via the HO-1/ROS signaling pathway. In this study, we demonstrated that Cur induced HO-1 expression and restored occludin and ZO-1 protein levels that were induced by SCI. Zinc protoporphyrin, which is a commonly used specific HO-1 inhibitor, reversed the protective role of Cur, suggesting that Cur has a protective effect that, at least in part, depends on an antioxidant mechanism based on HO-1 induction. In conclusion, our findings indicated that Cur effectively and doseindependently protected the BSCB through attenuation of inflammatory factors and TJ protein expression in the injured spinal cord. In addition, the effects of Cur on motor function after SCI were observed over the long term. However, Cur has been shown to inhibit the extrinsic and intrinsic pathways of blood coagulation and increase the chance of bleeding by inhibiting factor Xa and thrombin generation in human umbilical vein endothelial cells [39]. Therefore, the time and dosage of Cur should be carefully considered for each patient with SCI. More studies are essential to consider the possible therapeutic effects of Cur in these patients. There now appears to be new and potentially powerful opportunities for treating acute SCI by targeting the vascular responses. Conflict of interest We state that there is no conflict of interest. Acknowledgments This work was supported by Doctoral Scientific Research Starting Foundation of Liaoning Province, No. 20121096; Scientific Research Starting Foundation for PH.D. and Returned Overseas Teacher of Liaoning Medical University, No. Y2012B011; Program for Liaoning Excellent Talents in University, No. LR2013091; Special Funds for Clinical Medicine Construction of the Principal's Fund of the Liaoning Medical
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