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Neuroprotective effect of allicin in a rat model of acute spinal cord injury
Neuroprotective effect of allicin in a rat model of acute spinal cord injury Runxiao Lv, Ningfang Mao, Jinhui Wu, Chunwen Lu, Muchen Ding, Xiaochuan Gu, Yungang Wu, Zhicai Shi PII: DOI: Reference:
S0024-3205(15)30065-5 doi: 10.1016/j.lfs.2015.11.001 LFS 14548
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
3 June 2015 12 October 2015 2 November 2015
Please cite this article as: Lv Runxiao, Mao Ningfang, Wu Jinhui, Lu Chunwen, Ding Muchen, Gu Xiaochuan, Wu Yungang, Shi Zhicai, Neuroprotective effect of allicin in a rat model of acute spinal cord injury, Life Sciences (2015), doi: 10.1016/j.lfs.2015.11.001
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ACCEPTED MANUSCRIPT Neuroprotective effect of allicin in a rat model of acute spinal cord injury
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Runxiao Lv1, Ningfang Mao2, Jinhui Wu1, Chunwen Lu1, Muchen Ding1, Xiaochuan
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Unit of Graduate Students, Changhai Hospital of Second Military Medical University,
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Shanghai 200433, People’s Republic of China
Department of Spine Surgery, Changhai Hospital of Second Military Medical
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2
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Gu1, Yungang Wu1, Zhicai Shi2, *
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University, Shanghai 200433, People’s Republic of China
*
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Corresponding author: Dr. Zhicai Shi, Email: [email protected]. Department of
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Spine Surgery, Changhai Hospital of Second Military Medical University, 168
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Changhai Road, Shanghai 200433, People’s Republic of China
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ACCEPTED MANUSCRIPT Abstract Aims: This study aims to investigate the effect of allicin on motor functions and
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histopathologic changes after spinal cord injury and the mechanism underlying its
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neuroprotective effects.
Main methods: The motor function of rats was evaluated with the Basso, Beattie, and Bresna test. Histopathologic changes were evaluated by hematoxylin and eosin and
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Nissl staining. Spinal cord oxidative stress markers were determined by measuring
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glutathione and malondialdehyde content and superoxide dismutase activity using commercial kits. Inflammatory factors were determined by measuring tumor necrosis
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factor-α, interleukin-1β and interleukin-6 using ELISA assay. Apoptosis was
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examined using TUNEL staining. The effect of allicin on Nrf2 protein levels and
analysis.
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localization was assessed using immunofluorescence staining and western blotting
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Key findings: Results demonstrated that allicin accelerated the motor functional recovery and protected neuron damage against spinal cord injury (SCI). SCI-induced oxidative stress, inflammatory response and cell apoptosis in the spinal cord were also prevented by allicin. In addition, we observed that SCI increased Nrf2 nuclear expression, and allicin treatment further increased Nrf2 nuclear translocation in neurons and astrocytes. siRNA-mediated Nrf2 gene knockdown completely blocked the effect of allicin on spinal cord tissue. Significance: Our finding suggests that allicin promotes the recovery of motor function after SCI in rats, and this effect may be related to its anti-oxidant, 2
ACCEPTED MANUSCRIPT anti-inflammatory and anti-apoptotic effects. Allicin mediated Nrf2 nuclear
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translocation may be involved in the protective effect as well.
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Keywords: spinal cord injury, allicin, apoptosis, inflammation, oxidative stress, Nrf2
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ACCEPTED MANUSCRIPT 1. Introduction Acute traumatic spinal cord injury (SCI) usually results a catastrophic neural
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dysfunction which negatively affects the quality of life of patients. The biphasic injury
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process in SCI includes primary and secondary injury mechanisms. The primary injury is caused by the initial physical impact, which is characterized by acute bleeding and ischemia. Following the primary insult, several pathways trigger the
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secondary injury, causing reactive glial changes, neuronal inflammation, oxidative
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stress, neuronal degeneration and apoptosis [1-4]. Even worse, the spontaneous anatomical and functional recovery will be prevented by these subsequent
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pathological events. Unfortunately, primary injury caused neuronal loss cannot be
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restored. Thus, most of the attention has been paid on the development of therapeutic
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strategy for the secondary injury. However, despite numerous promising experimental studies have been reported, effective treatment that can overcome secondary damage
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after SCI is lacking.
The nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is a classical signal that responsible for the cellular redox homeostasis. Activating this pathway is one of the main defense mechanisms against oxidative stress [5-7] and has been reported to be neuroprotective after SCI [8]. In SCI rats, Nrf2 levels were peaked at 30 min after SCI in cytoplasmic fractions and remained elevated for 3 days, and levels in nuclear fractions elevated at 30 min and peaked at 6 h following SCI. In addition, pharmacological activation of Nrf2/ARE pathway may contribute to the locomotion recovery and inhibit inflammatory 4
ACCEPTED MANUSCRIPT response [8, 9]. Allicin (diallyl thiosulfinate), is a small molecule extracted from the garlic. It is
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known as one of the most biologically active compounds in garlic, which is
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responsible for most of the functions of garlic [10]. Numerous studies have demonstrated the pharmacological effect of allicin, including anti-inflammatory, antimicrobial, antifungal, antiparasitic, antihypertensive, anti-diabetic and anti-tumor
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activities [11, 12]. Recent in vitro and in vivo studies reported that allicin could
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prevent traumatic neuronal injury in rat cortical neurons and traumatic brain injury rats by attenuating oxidative stress, inhibiting inflammatory response and neuron
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apoptosis [13, 14]. In addition, allicin has been found to have neuroprotective effect
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against spinal cord ischemia/reperfusion injury in rabbits [15]. However, its effect on
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acute traumatic spinal cord injury has not been investigated. In addition, Nrf2 activation has been approved to be beneficial in the recovery of SCI [16, 17]. In a
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previous study, allicin has been found to active Nrf2 signaling pathway in cardiac hypertrophy rats [18]. Therefore, we hypothesize the allicin would have neuroprotective effect via Nrf2 activation and aim to prove it in this study.
2. Material and Methods 2.1. Establishment of animal models of spinal cord injury A total of 90 female Sprague-Dawley rats weighing 200–250 g were purchased from Shanghai SLAC Laboratory Animal Co (Shanghai, China). All the animal experiments were performed in accordance with the guidelines of the Ethical 5
ACCEPTED MANUSCRIPT Committee of Experimental Animals of Second Military Medical University. After being acclimatized for 2 weeks, rats were randomly divided into the following groups
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(n=18): (1) sham; (2) SCI; (3) SCI+allicin 2mg/kg (allicin L); (4) SCI+allicin
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10mg/kg (allicin M); (5) SCI+allicin 50mg/kg (allicin H). All the rats were intraperitoneally anesthetized with 30 mg/kg pentobarbital sodium. The lower back was shaved and sterilized. A median incision was made on the back taking T8–9
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spinous process as a center to expose T7–10 spinous processes and the lamina and a
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laminectomy was performed at the T8 level. Rats in the sham group received only T8 laminectomy. Rats in the SCI groups received an injury in accordance with Allen's
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method. Briefly, a self-made 10 g rod was dropped vertically from a height of 50 mm
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on the exposed spinal cord. The rod was rest on the injury site for 3 min. The wound
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was then washed and the tissue was sutured. NaCl (5 mL of 0.9%) was injected intraperitoneally immediately after surgery, and penicillin (200,000 units/day,
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intramuscular injection) was used to protect from postoperative infection. Urinary bladder was manually emptied twice daily to assist in urination until the micturition function returned to normal. Rats in allicin groups received an intraperitoneally injection of allicin (MB5783, Meilun, Dalian, China) one hour before the surgery and once daily for 21 days. Rats in the sham and SCI groups received 0.9% NaCl daily.
2.2. siRNA design and delivery siRNAs encoding Nrf2 were purchased from GenePharma (Shanghai, China). Sequence
of
the
siRNA
used
in
the 6
present
study
is
as
followed:
ACCEPTED MANUSCRIPT CCGGAGAAAUUCCUCCCAAUTT. The negative scrambled sequence is as follows: UUCUCCGAACGUGUCACGUTT. After surgery, a hole was created at the exposed
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dura using a hypodermic syringe. An intrathecal catheter filled with sterile PBS was
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inserted 7 mm cephalad into the dura. The catheter exposed epidural was fixed tightly on the surrounding soft tissue. The end of the catheter was fixed on the skin and was sealed sterilely. Rats in siRNA treated groups received daily 5 μg/5 μl of Nrf2-siRNA
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or scramble siRNA through the intrathecal catheter for 3 days successively. The
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mRNA and protein expression of Nrf2 in the spinal cord was measured by real-time
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PCR and western blot, respectively.
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2.3.Evaluation of motor function
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Six rats in each group were randomly selected to perform motor function test at day 1, 4, 7, 14 and 21 after the surgery. Motor function was assessed using the Basso, Beattie
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and Bresnahan (BBB) locomotor rating scale [19]. The range of BBB locomotor scale scores was between 0 and 21. Twenty-one points refer to normal motor capacity and lower scores indicate impairment in motor capacity. Each rat was evaluated three times by three observers who were blinded to the treatment group, and the mean of the three measurements was taken.
2.4.Histological examinations After the 21-day treatment, six rats in each group were deeply anesthetized and perfused with 4% paraformaldehyde via the left ventricle for pre-fix. A 1.5 cm spinal 7
ACCEPTED MANUSCRIPT cord tissue surrounding the damage site was collected and post-fixed in 4% paraformaldehyde overnight. Fixed tissues were dehydrated using a series of ethanol,
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embedded in paraffin, and serially sectioned into 4 μm thick coronal slices. To
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perform staining, sections were deparaffinized in xylene and hydrated using a series of ethanol. Three sections of each group were used for hematoxylin and eosin (HE) (Solarbio Science & Technology, Beijing, China) staining. Three sections were
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stained with thionine (Solarbio). Five fields within each slide were randomly selected
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for neuronal counts. Sections were viewed under an optical microscope (DP73; Olympus, Tokyo, Japan). The cavity area was measured on sections stained with HE
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using IMAGE J software (National Institutes of Health, Bethesda, MD, USA).
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2.5.Tissue preparation and protein quantification After the 21-day treatment, the remained 6 rats in each group were deeply
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anesthetized and spinal cord tissue surrounding the damage site was immediately collected. Tissues were homogenized by a homogenizer in cooled RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) on ice. Homogenates were then centrifuged at 15,000 g for 10 min at 4°C. Nuclear and cytosolic proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) following the manufacturer’s instruction. The protein concentrations of the supernatants were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime).
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ACCEPTED MANUSCRIPT 2.6.Biochemical determination Spinal cord tissues were homogenized in cooled PBS and repeated freezing in liquid
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nitrogen and thawing for three times. Homogenates were then centrifuged at 15,000 g
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for 10 min at 4°C. Supernatants were used for the measurement of MDA and GSH levels and determination of SOD activity according to the protocols in the commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing,
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China).
2.7.Enzyme-linked immunosorbent assay (ELISA)
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Spinal cord protein supernatants were prepared in RIPA as described below. The
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determination of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 were
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performed using commercially available ELISA kits special for rats (USCN Life Science, Wuhan, China) according to the manufacturer’s instructions. Concentrations
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are given in pg/mg protein.
2.8.Real-time quantitative PCR Total RNA from tissue specimens in each group were purified using an RNA simple Total RNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's protocol. Oligonucleotide primer and super Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV) (BioTeke, Beijing, China) were used to make cDNA in a 20-μL reaction mixture. Quantitative real-time PCR was performed on 1-μL cDNA using 10-μL SYBR-Green Master Mix (Tiangen Biotechnology Co., Ltd., Beijing, 9
ACCEPTED MANUSCRIPT China), 1-μL cDNA and 10 μM of forward and reverse primers on an Exicycler™ 96 real-time quantitative thermal block (Bioneer, Daejeon, Korea) in a 20-μL reaction
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CTGGAATGGAAGGAGATGCC-3’, reverse: 5’-
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mixture. The sequences of primers are as follows: HO-1, forward: 5’-
TCAGAACAGCCGCCTCTACCG-3’, and β-actin, forward: 5’GGAGATTACTGCCCTGGCTCCTAGC-3’, reverse: 5’-
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GGCCGGACTCATCGTACTCCTGCTT-3’ (Sangon Biotech, Shanghai, China). The
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reaction was performed in an Exicycler™ 96 real-time quantitative thermal block
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(Bioneer, Taejon, Korea). Data were analyzed by using the 2−ΔΔCt method.
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2.9.Double immunofluorescence staining
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Dewaxed sections were incubated in Tris-Buffered saline (PBS) containing 0.1% Triton X-100 for 30 min and blocked with goat serum (Beyotime) for 30 min at room
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temperature. After being washed with PBS, sections were incubated with a mouse monoclonal anti-NeuN antibody (1:100, ab104224, Abcam, Cambridge, MA, USA) or a mouse monoclonal anti-GFAP antibody (1:50, sc-33673, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and a rabbit polyclonal anti-Nrf2 antibody (1:100, bs-1074R, Bioss, Beijing, China) overnight at 4°C. Sections were washed and then incubated in FITC labeled goat anti-mouse secondary antibody (A5608, 1:200, Beyotime) and Cy3 labeled goat anti-rabbit secondary antibody (A0516, 1:200, Beyotime), each for 1 hour at 37 °C in darkness. Sections were mounted in aqueous mounting medium (Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China). Fluorescent 10
ACCEPTED MANUSCRIPT labeling was examined using a confocal system (Olympus, Tokyo, Japan).
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Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling
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2.10.
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(TUNEL)
Staining was performed using a TUNEL apoptosis detection kit (Wuhan Boster Biological Engineering Co., Ltd., Wuhan, China), according to the manufacturer's
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instruction. Sections were counterstained with haematoxylin (Solarbio) and mounted
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using Permount. Apoptotic cells that appeared brown under a microscope (DP73;
Western blot analysis
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2.11.
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Olympus) were counted in five fields of view per section, at 400× magnification.
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Equal amounts of protein were heat-denatured and loaded on a 12% sodium dodecyl sulfate-polyacrylamide gel to perform electrophoresis. Separated proteins were then
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transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using a wet transfer method. The membrane was blocked for 1 hour at room temperature with PBS containing 5% skimmed milk and 0.1% Tween-20, washed with 1 × Tris Buffered Saline, with Tween-20 (TBST) for three times and then incubated overnight at 4°C with primary antibodies specific for β-actin (1:1000, sc-47778, mouse monoclonal, Santa Cruz Biotechnology), Bcl-2 (1:400, BA0412, rabbit polyclonal, Boster, Wuhan, China), Bax (1:400, rabbit polyclonal, BA0315, Boster), cleaved-caspase 3 (1:500, bs-0081R, rabbit polyclonal, Bioss), Nrf2 (1:500, bs-1074R, rabbit polyclonal, Bioss), HO-1 (1:200, sc-10789, rabbit polyclonal, Santa 11
ACCEPTED MANUSCRIPT Cruz Biotechnology), and Histone H3 (1:1000, bs-17422R, rabbit polyclonal, Bioss) in the blocking buffer. The membrane was subsequently incubated with HRP
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conjugated secondary antibodies (1:5000; Beyotime), then visualized using an
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enhanced chemiluminescence system (7 Sea Pharmtech, Shanghai, China) and exposed on Fuji Rx 100 X-ray film (Fuji Photo Film, Tokyo, Japan). Bands were analyzed using Gel-Pro-Analyzer (Media Cybernetics, Bethesda, MD, USA). β-actin
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2.12.
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was taken as an loading control.
Statistical analysis
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All values are presented as the mean ± SD. Statistical analysis was performed using
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SPSS version 19.0 software (IBM, New York, NY, USA). The BBB scores were
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analyzed using Kruskal-Wallis test. Other data were analyzed using one-way analysis of variance and the least significant difference test. P value less than 0.05 was
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considered statistical significance.
3. Results
3.1. BBB test Rats were subjected to BBB test to evaluate the effect of allicin on functional improvements at 1, 4, 7, 14 and 21 days after surgery (Figure 1). The BBB score of rats in the sham group was 21, which indicated a normal motor function. One day after surgery, the motor function of rats was obviously impaired, represented by the dramatically decreased BBB scores. However, 14 days after allicin treatment, the 12
ACCEPTED MANUSCRIPT BBB score of rats in allicin 50 mg/kg group were significantly higher than that in the SCI group (P<0.05). At day 21, the score in allicin 50 mg/kg group reached higher
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(P<0.01 vs. SCI group) and allicin 10 mg/kg showed effective in improving motor
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functional recovery on SCI rats (P<0.05 vs. SCI group). There was not any significant difference between mean BBB scores of allicin 2 mg/kg and SCI groups (P>0.05).
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3.2. Effect of allicin on neuron damage in the spinal cords of SCI rats
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Neuron survival in the spinal cord was evaluated by HE and Nissl staining. Twenty-one days after surgery, marked necrosis, edema and loss of Nissl granules
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were observed in SCI groups (Figure 3). In the allicin 10 and 50 mg/kg treated groups,
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retained neurons with improved morphology and increased Nissl bodies were
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observed. These results indicate that allicin intervention may improve the histological
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changes of spinal cord and restore neuron function after SCI.
3.3.Effect of allicin on oxidative stress status in the spinal cords of SCI rats Oxidative stress status was examined by MDA level, GSH level and SOD activity assays. Following SCI, spinal cord tissue MDA level increased, and GSH level and SOD activity decreased significantly when the sham groups were compared with the SCI group (Figure 4, P < 0.01). Treatment with 10 and 50 mg/kg allicin significantly decreased the tissue MDA level and increased the tissue GSH level and SOD activity compared with the SCI group (P < 0.01). There was no significant difference between the 2 mg/kg allicin treatment and SCI groups though 2 mg/kg also decreased the mean 13
ACCEPTED MANUSCRIPT of those indexes (P > 0.05).
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3.4.Effect of allicin on inflammatory responses in the spinal cords of SCI rats
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Typical cytokines associated with inflammation (TNF-α, IL-1β and IL-6) in the spinal cord of each group 21-day post-injury were detected using ELISA. Levels of all the three cytokines were dramatically increased in the SCI group (Figure 5, P < 0.01).
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Allicin dose-dependently decreased these inflammatory cytokines secretion. Levels of
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TNF-α and IL-6 could be significantly decreased by 10 and 50 mg/kg allicin, and the
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level of IL-1β could be significantly decreased by all the 3 dosages of allicin.
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3.5.Effect of allicin on apoptosis in the spinal cords of SCI rats
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The cell apoptosis in the spinal lesions were detected by TUNEL staining. Sham group showed no apoptosis positive cells (Figure 5A). The numbers of
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TUNEL-positive cells increased significantly 21-day post-injury (Figure 5B, P<0.01 vs. sham group). Allicin dose-dependently decreased TUNEL-positive cells in the spinal cord (Figure 5C-E). In addition, expressions of apoptosis-associated proteins Bcl-2, Bax and cleaved-caspase 3 were also detected by western blot. In consistent with the result of TUNEL staining, allicin dose-dependently upregulated the expression of Bcl-2 and downregulated the expression of Bax and cleaved-caspase 3 (Figure 5H-K). Both of 10 and 50 mg/kg allicin showed the significant anti-apoptotic effect, whereas 2 mg/kg allicin has not shown significant effect.
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ACCEPTED MANUSCRIPT 3.6.Effect of allicin on Nrf2 nuclear translocation in the spinal cords of SCI rats The expression and nuclear translocation were detected using immunofluorescence
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staining and western blot. As shown in Figure 6A, following the surgery, Nrf2 protein
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showed an obvious nuclear translocation in both of the neuron and astrocyte, which was consistent with the previous study [8]. In addition, after allicin treatment, nuclear expression of Nrf2 was increased in 10 and 50 mg/kg allicin groups. The results of
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western blot analysis confirmed the observation in immunofluorescence staining.
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Nuclear expression of Nrf2 and expression of HO-1 was markedly upregulated by SCI and further upregulated by allicin 10 and 50 mg/kg treatment (Figure 6C-E). These
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impaired spinal cord.
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results suggested that allicin may promote Nrf2 nuclear translocation in the traumatic
3.7.Blockage of Nrf2 inhibits the protective effect of allicin
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In order to further study the role of Nrf2 in the protective effect of allicin, the siRNA-mediated gene knockdown was used. As illustrated in Figure 7, Nrf2-siRNA acute intrathecal injection successfully inhibited expression of Nrf2 in spinal cord tissue. In addition, Nrf2-siRNA intrathecal injection completely blocked the effect of allicin on SCI rats. The BBB scores were significantly lower than that in the allicin therapy group (Figure 8A). The scramble siRNA injection has not affected the therapeutic effect of allicin. In addition, Nrf2-siRNA also blocked the antioxidant effect of allicin. The level of MDA was significantly increased (Figure 8B) and level of GSH and activity of SOD were decreased (Figure 8C and D). These data suggested 15
ACCEPTED MANUSCRIPT that allicin protected rats from SCI through Nrf2 signal activation.
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4. Discussion
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In the present study, we demonstrated that allicin could improve motor function recovery of SCI rats, dose-dependently decreased neuronal death, attenuate oxidative stress and inflammatory response, and inhibite cell apoptosis in the lesion site of
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spinal cord. In addition, allicin mediated activation of Nrf2 signal may be involved in
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the protective effect of allicin.
Allicin has been demonstrated to have neuroprotective effects in some disease
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including neurodegenerative diseases and neurotrauma. In the present study, the
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neuroprotective effects of allicin were evaluated on SCI rats, which had not been
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previously studied. In the histopathological examination, acute SCI caused large cavity, hemorrhage, edema, and necrosis in the spinal cord. Additionally, in the SCI
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group, neurons with normal shape and function in the spinal cord were significantly decreased in number. This neuronal damage was consistent with the impaired motor function. Spinal cords of rats in both 10 and 50 mg/kg allicin groups showed better histological results and a high number of normal neurons compared with the SCI group. These two dosages of allicin treatment also improved motor function recovery in SCI rats at 21-day post-surgery. These results indicate that allicin has neuroprotective effect on spinal cord of SCI rats, which may contribute to the recovery of motor function. Primary and secondary injuries of acute spinal cord injury lead to neuronal death 16
ACCEPTED MANUSCRIPT in spinal cord. Among the secondary injury factors, oxidative stress, inflammation and apoptosis are the most important and have been paid most attention [20-25].
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Oxidative stress following SCI produces reactive oxygen species (ROS) and initiates
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lipid peroxidation activity in the injured neural tissue [26]. ROS leads damages to lipids, proteins and nucleic acids, and subsequently causes cytotoxicity [27]. The central nervous system is highly vulnerable to oxidative injury because the neural
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tissues consist largely of lipids. Previous studies demonstrated that allicin decreased
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reactive oxygen species (ROS) generation, reduced lipid peroxidation and increased antioxidant enzyme activities in various tissues including liver, kidney and spinal cord
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[28, 29]. In the present study, allicin inhibited oxidative stress by decreasing MDA
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level and increasing GSH level and SOD activity in a dose dependent manner, which
of allicin.
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indicates that the anti-oxidative effect may be involved in the neuroprotective effect
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Inflammatory responses play an important role in the secondary injury after SCI [30]. Elevated expressions of inflammatory cytokines have been demonstrated at both the mRNA and protein levels in spinal cords of animal models of SCI [31-33]. These cytokines are involved in neuronal necrosis and apoptosis, leucocyte infiltration and activation of glial cells, and play an important role in neurodegeneration in SCI [34, 35]. In the present study, 21 days after SCI, levels of TNF-α, IL-1β and IL-6 were significantly increased in the spinal cord. Allicin treatment markedly reduced the production of these inflammatory factors in a dose-dependent manner. This result suggests that the anti-inflammatory effect may be involved in the neuroprotective 17
ACCEPTED MANUSCRIPT effect of allicin. Acute SCI caused apoptosis leads to death of neuronal cells in the spinal cord,
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especially neurons and oligodendrocytes. This will further destroy the axon-myelin
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structure and impair impulse conduction, resulting in neuronal dysfunction [36, 37]. Neuronal apoptosis initiates as early as 4 hours around the lesion site after SCI, and neuronal and oligodendroglial apoptosis may last for weeks [38-40]. According to the
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previous studies, allicin has a biphasic effect on cell apoptosis. It has been
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demonstrated to induce apoptosis in numerous kinds of cancer cells [41-44]. However, it has also been shown to have anti-apoptotic effect on other diseases [45, 46]. In the
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present study, allicin effectively reduced number of apoptosis cells in the injured
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spinal cord tissue, upregulated the expression of Bcl-2 and downregulated expressions
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of Bax and cleaved-caspase 3, which are responsible for the mitochondrial pathway apoptosis. This result indicates the anti-apoptotic effect of allicin may contribute to its
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neuroprotective effect in SCI rats. The activation of Nrf2/ARE signal is dependent
upon the nuclear translocation
of Nrf2, which subsequently induces the transcriptions of protective genes [47]. In agreement with this theory, our results showed that SCI induces increased expression of Nrf2 in the nuclear of neurons and astrocytes. Allicin treatment after SCI further enhanced expression of the Nrf2 nuclear translocation resulting in increased expression of the HO-1. In response to oxidative stress, Nrf2 induces expressions of a number of antioxidant genes and other cytoprotective phase II detoxifying enzymes, such as 18
ACCEPTED MANUSCRIPT HO-1, SOD, glutathione peroxidase (GPx) and glutathione-S-transferases. Numerous studies have shown that Nrf2 knockout mice underwent highly susceptible to
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cytotoxic compounds compared to wild-type mice due to the decreased levels of
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antioxidant proteins and phase II detoxification enzymes [48-51]. In addition to anti-oxidant effect, overexpression of Nrf2 is also considered to protect cells from apoptosis and inflammatory response [52-54]. Our observation showed that the
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neuroprotective effect of allicin on SCI rats is Nrf2 activation-dependent, siRNA
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mediated Nrf2 gene knockdown completely blocked the effect of allicin, which indicated that allicin protected spinal cord tissue from traumatic injury through Nrf2
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activation.
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In this study, all the experiments were performed on female rats. It is well known
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that the menstrual cycle may affect some biological processes like inflammatory cytokines production, and the hormone may even have therapeutic effect on various
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disorders including SCI [55]. Although the menstrual cycle of animals has not been synchronized in this study, we found the data from each animal did not fluctuate much and the dose-dependent effect of allicin is remarkably. Thus we believe that the effect of menstrual cycle on our study is small and the neuroprotective effect seen in the SCI rats was come from allicin. 5. Conclusion In conclusion, the present study reveals that allicin exhibits neuroprotective effects through anti-apoptotic, anti-inflammatory, and anti oxidant effects on the injury spinal cord of rats. This effect is Nrf2 activation-dependent. This study 19
ACCEPTED MANUSCRIPT provides a novel neuroprotective drug candidate for SCI therapy.
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Conflict of interest statement
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The authors declare that they have no conflicts of interest concerning this article.
References
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[1] Block, Zecca, Hong, Microglia-mediated neurotoxicity: uncovering the molecular
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mechanisms, Nat Rev Neurosci 8 (2007) 57-69.
[2] Fleming, Norenberg, Ramsay, Dekaban, Marcillo, Saenz, et al., The cellular
D
inflammatory response in human spinal cords after injury, Brain 129 (2006)
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3249-3269.
89-105.
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[3] Graeber, Streit, Microglia: biology and pathology, Acta Neuropathol 119 (2010)
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[4] Yiu, He, Glial inhibition of CNS axon regeneration, Nat Rev Neurosci 7 (2006) 617-627.
[5] McWalter, Higgins, McLellan, Henderson, Song, Thornalley, et al., Transcription factor Nrf2 is essential for induction of NAD(P)H:quinone oxidoreductase 1, glutathione S-transferases, and glutamate cysteine ligase by broccoli seeds and isothiocyanates, J Nutr 134 (2004) 3499S-3506S. [6] Alam, Stewart, Touchard, Boinapally, Choi, Cook, Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene, J Biol Chem 274 (1999) 26071-26078. 20
ACCEPTED MANUSCRIPT [7] Chanas, Jiang, McMahon, McWalter, McLellan, Elcombe, et al., Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression
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of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes
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in the livers of male and female mice, Biochem J 365 (2002) 405-416. [8] Wang, de Rivero Vaccari, Wang, Diaz, German, Marcillo, et al., Activation of the nuclear factor E2-related factor 2/antioxidant response element pathway is
NU
neuroprotective after spinal cord injury, J Neurotrauma 29 (2012) 936-945.
MA
[9] Jin, Wang, Zhu, Yuan, Ni, Jiang, et al., Anti-inflammatory effects of curcumin in experimental spinal cord injury in rats, Inflamm Res 63 (2014) 381-387.
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[10] Lawson, Gardner, Composition, stability, and bioavailability of garlic products
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used in a clinical trial, J Agric Food Chem 53 (2005) 6254-6261.
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[11] Hunter, Caira, Stellenboom, Thiolsulfinate allicin from garlic: inspiration for a new antimicrobial agent, Ann N Y Acad Sci 1056 (2005) 234-241.
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[12] Bayan, Koulivand, Gorji, Garlic: a review of potential therapeutic effects, Avicenna J Phytomed 4 (2014) 1-14. [13] Zhou, Li, Han, Gao, He, Li, et al., Allicin protects rat cortical neurons against mechanical trauma injury by regulating nitric oxide synthase pathways, Brain Res Bull 100 (2014) 14-21. [14] Chen, Qi, Feng, Wang, Bao, Wang, et al., Neuroprotective effect of allicin against traumatic brain injury via Akt/endothelial nitric oxide synthase pathway-mediated anti-inflammatory and anti-oxidative activities, Neurochem Int 68 (2014) 28-37. [15] Zhu, Chen, Guan, Liu, Liu, Neuroprotective effects of allicin on spinal cord 21
ACCEPTED MANUSCRIPT ischemia-reperfusion injury via improvement of mitochondrial function in rabbits, Neurochem Int 61 (2012) 640-648.
IP
T
[16] Wang, Shen, Xie, Ling, Lu, Neuroprotective effect of ginseng against spinal cord
SC R
injury induced oxidative stress and inflammatory responses, Int J Clin Exp Med 8 (2015) 3514-3521.
[17] Jin, Ni, Hou, Ming, Wang, Yuan, et al., Tert-butylhydroquinone protects the spinal
NU
cord against inflammatory response produced by spinal cord injury, Ann Clin Lab Sci
MA
44 (2014) 151-157.
[18] Li, Li, Xiang, Hu, Lu, Tian, et al., Allicin ameliorates cardiac hypertrophy and
TE
Ther 26 (2012) 457-465.
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fibrosis through enhancing of Nrf2 antioxidant signaling pathways, Cardiovasc Drugs
CE P
[19] Basso, Beattie, Bresnahan, A sensitive and reliable locomotor rating scale for open field testing in rats, J Neurotrauma 12 (1995) 1-21. Gul,
Bektas,
Hanci,
Acikgoz,
Effects
of
dexmedetomidine
or
AC
[20] Can,
methylprednisolone on inflammatory responses in spinal cord injury, Acta Anaesthesiol Scand 53 (2009) 1068-1072. [21] Beck, Nguyen, Galvan, Salazar, Woodruff, Anderson, Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment, Brain 133 (2010) 433-447. [22] Nakajima, Osuka, Seki, Gupta, Hara, Takayasu, et al., Taurine reduces inflammatory responses after spinal cord injury, J Neurotrauma 27 (2010) 403-410. 22
ACCEPTED MANUSCRIPT [23] Dinc, Iplikcioglu, Atabey, Eroglu, Topuz, Ipcioglu, et al., Comparison of deferoxamine and methylprednisolone: protective effect of pharmacological agents on
IP
T
lipid peroxidation in spinal cord injury in rats, Spine (Phila Pa 1976) 38 (2013)
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E1649-1655.
[24] Vaziri, Lee, Lin, Lin, Sindhu, NAD(P)H oxidase, superoxide dismutase, catalase,
injury, Brain Res 995 (2004) 76-83.
NU
glutathione peroxidase and nitric oxide synthase expression in subacute spinal cord
Prog Brain Res 137 (2002) 37-47.
MA
[25] Beattie, Hermann, Rogers, Bresnahan, Cell death in models of spinal cord injury,
D
[26] Torres, Salgado-Ceballos, Torres, Orozco-Suarez, Diaz-Ruiz, Martinez, et al.,
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Early metabolic reactivation versus antioxidant therapy after a traumatic spinal cord
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injury in adult rats, Neuropathology 30 (2010) 36-43. [27] Dawson, Dawson, Snyder, Molecular mechanisms of nitric oxide actions in the
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brain, Ann N Y Acad Sci 738 (1994) 76-85. [28] Abdel-Daim, Abdelkhalek, Hassan, Antagonistic activity of dietary allicin against deltamethrin-induced oxidative damage in freshwater Nile tilapia; Oreochromis niloticus, Ecotoxicol Environ Saf 111 (2015) 146-152. [29] Liu, Ren, Wang, Yao, He, Allicin protects spinal cord neurons from glutamate-induced oxidative stress through regulating the heat shock protein 70/inducible nitric oxide synthase pathway, Food Funct 6 (2015) 321-330. [30] Hayashi, Ueyama, Nemoto, Tamaki, Senba, Sequential mRNA expression for immediate early genes, cytokines, and neurotrophins in spinal cord injury, J 23
ACCEPTED MANUSCRIPT Neurotrauma 17 (2000) 203-218. [31] Wu, Yang, Mei, Yu, Selective inhibition of STAT1 reduces spinal cord injury in
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mice, Neurosci Lett 580 (2014) 7-11.
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[32] Guo, Li, Shi, Wang, Yuan, Shi, et al., Genetic ablation of receptor for advanced glycation end products promotes functional recovery in mouse model of spinal cord injury, Mol Cell Biochem 390 (2014) 215-223.
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[33] Jiang, Tu, Zou, Hu, Wang, Li, et al., Neuroprotective effects of different
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modalities of acupuncture on traumatic spinal cord injury in rats, Evid Based Complement Alternat Med 2014 (2014) 431580.
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[34] Lee, Yune, Baik, Shin, Du, Rhim, et al., Role of tumor necrosis factor-alpha in
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neuronal and glial apoptosis after spinal cord injury, Exp Neurol 166 (2000) 190-195.
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[35] Hurst, Wilkinson, McLoughlin, Jones, Horiuchi, Yamamoto, et al., Il-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment
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seen during acute inflammation, Immunity 14 (2001) 705-714. [36] Crowe, Bresnahan, Shuman, Masters, Beattie, Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys, Nat Med 3 (1997) 73-76. [37] Pannu, Barbosa, Singh, Singh, Attenuation of acute inflammatory response by atorvastatin after spinal cord injury in rats, J Neurosci Res 79 (2005) 340-350. [38] Liu, Xu, Hu, Du, Zhang, McDonald, et al., Neuronal and glial apoptosis after traumatic spinal cord injury, J Neurosci 17 (1997) 5395-5406. [39] Casha, Yu, Fehlings, Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat, 24
ACCEPTED MANUSCRIPT Neuroscience 103 (2001) 203-218. [40] Shuman, Bresnahan, Beattie, Apoptosis of microglia and oligodendrocytes after
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spinal cord contusion in rats, J Neurosci Res 50 (1997) 798-808.
gastric
carcinoma
cell
line
through
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[41] Zhang, Zhu, Duan, Feng, He, Allicin induces apoptosis of the MGC-803 human the
p38
mitogen-activated
protein
kinase/caspase-3 signaling pathway, Mol Med Rep 11 (2015) 2755-2760.
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[42] Chu, Ho, Chung, Raghu, Lo, Sheen, Allicin induces anti-human liver cancer cells
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through the p53 gene modulating apoptosis and autophagy, J Agric Food Chem 61 (2013) 9839-9848.
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[43] Xu, Yu, Zhai, Zhang, Shen, Bai, et al., Role of JNK Activation and Mitochondrial
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Bax Translocation in Allicin-Induced Apoptosis in Human Ovarian Cancer SKOV3
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Cells, Evid Based Complement Alternat Med 2014 (2014) 378684. [44] Cha, Choi, Cha, Choi, Cho, Allicin inhibits cell growth and induces apoptosis in
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U87MG human glioblastoma cells through an ERK-dependent pathway, Oncol Rep 28 (2012) 41-48.
[45] Chen, Tang, Qian, Chen, Zhang, Wo, et al., Allicin prevents H(2)O(2)-induced apoptosis of HUVECs by inhibiting an oxidative stress pathway, BMC Complement Altern Med 14 (2014) 321. [46] Liu, Qi, Wang, Wu, Cao, Huang, et al., Allicin protects against myocardial apoptosis and fibrosis in streptozotocin-induced diabetic rats, Phytomedicine 19 (2012) 693-698. [47] Liu, Li, Bu, Li, Li, Sun, et al., The neuroprotective potential of phase II enzyme 25
ACCEPTED MANUSCRIPT inducer on motor neuron survival in traumatic spinal cord injury in vitro, Cell Mol Neurobiol 28 (2008) 769-779.
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[48] Mao, Wang, Qiao, Wang, Disruption of Nrf2 enhances the upregulation of nuclear
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factor-kappaB activity, tumor necrosis factor-alpha, and matrix metalloproteinase-9 after spinal cord injury in mice, Mediators Inflamm 2010 (2010) 238321. [49] Ramos-Gomez, Kwak, Dolan, Itoh, Yamamoto, Talalay, et al., Sensitivity to
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carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in
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nrf2 transcription factor-deficient mice, Proc Natl Acad Sci U S A 98 (2001) 3410-3415.
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[50] Aoki, Sato, Nishimura, Takahashi, Itoh, Yamamoto, Accelerated DNA adduct
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formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust, Toxicol
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Appl Pharmacol 173 (2001) 154-160. [51] Enomoto, Itoh, Nagayoshi, Haruta, Kimura, O'Connor, et al., High sensitivity of
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Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes, Toxicol Sci 59 (2001) 169-177. [52] Kotlo, Yehiely, Efimova, Harasty, Hesabi, Shchors, et al., Nrf2 is an inhibitor of the Fas pathway as identified by Achilles' Heel Method, a new function-based approach to gene identification in human cells, Oncogene 22 (2003) 797-806. [53] Li, Khor, Xu, Shen, Jeong, Yu, et al., Activation of Nrf2-antioxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis, Biochem Pharmacol 76 (2008) 1485-1489. 26
ACCEPTED MANUSCRIPT [54] Peng, Geh, Chen, Meng, Fan, Sartor, et al., Inhibitor of kappaB kinase beta regulates redox homeostasis by controlling the constitutive levels of glutathione, Mol
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Pharmacol 77 (2010) 784-792.
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[55] Cox, Varma, Barry, Vertegel, Banik, Nanoparticle Estrogen in Rat Spinal Cord Injury Elicits Rapid Anti-Inflammatory Effects in Plasma, Cerebrospinal Fluid, and (2015).
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Tissue, J Neurotrauma
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ACCEPTED MANUSCRIPT Figure legends Figure 1 Influence of administration of allicin on the motor function of rats in
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21-day period of study. Allicin (10 and 50 mg/kg) treatment improved motor
± SD (6 rats per groups).
##
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functional recovery in SCI rats at day 21 post-injury. The data are presented as mean : P < 0.01 compared with sham group. *: P < 0.05
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compared with SCI group. **: P < 0.01 compared with SCI group.
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Figure 2 Influence of administration of allicin on spinal cord neurons. Haematoxylin and eosin and (A-E) Nissl staining (F-K) was performed to evaluate the
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effect of allicin on neurons in the spinal cord. (A and F) sham group, (B and G) SCI
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group, (C and H), SCI+allicin 2 mg/kg group, (D and I) SCI+allicin 10 mg/kg group,
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(E and J) SCI+allicin 50 mg/kg group, and (K) quantification of number of Nissl bodies. Scale bar: 100 μm. The data are presented as mean ± SD (6 rats per groups). ##:
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P < 0.01 compared with sham group. **: P < 0.01 compared with SCI group.
Figure 3 Influence of administration of allicin on the status of oxidative stress in the spinal cord of SCI rats. (A) Malondialdehyde (MDA) level, (B) glutathione (GSH) and (C) superoxide dismutase (SOD) activity. Allicin dose-dependently attenuated oxidative stress in the spinal cord. The data are presented as mean ± SD (6 rats per groups). ##: P < 0.01 compared with sham group. **: P < 0.01 compared with SCI group.
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ACCEPTED MANUSCRIPT Figure 4 Influence of administration of allicin on the inflammatory response in the spinal cord of SCI rats. Allicin dose-dependently decreased tumor necrosis
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factor-α (TNF-α), interleukin (IL)-1β and IL-6 secretion in the spinal cord of SCI rats.
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The data are presented as mean ± SD (6 rats per groups). ##: P < 0.01 compared with sham group. **: P < 0.01 compared with SCI group.
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Figure 5 Influence of administration of allicin on cell apoptosis in the spinal cord
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of SCI rats. Effect of allicin on cell apoptosis was evaluated using TUNEL staining (A-F) and western blot (G-J). (A) sham group, (B) SCI group, (C), SCI+allicin 2
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mg/kg group, (D) SCI+allicin 10 mg/kg group, (E) SCI+allicin 50 mg/kg group, and
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(F) quantification of apoptosis cells. (G) Blots of apoptotic-associated proteins, (H-J)
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semi-quantifications of protein levels. Allicin dose-dependently attenuated cell apoptosis in the spinal cord of SCI rats. Scale bar: 50 μm. The data are presented as
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mean ± SD (6 rats per groups). ##: P < 0.01 compared with sham group. **: P < 0.01 compared with SCI group.
Figure 6 Influence of administration of allicin on Nrf2 nuclear translocation in the spinal cord of SCI rats. (A) Double immunofluorescence staining of Nrf2 and NeuN or GFAP. Nrf2 immunoreactivity increases after SCI and allicin administration in neurons and astrocytes of the spinal cord. (B) Protein expression of cytoplasmic Nrf2, (C) protein expression of nuclear Nrf2, (D) protein expression of HO-1, and (E) mRNA expression of HO-1 in the spinal cord. Scale bar: 50 μm in NeuN staining 29
ACCEPTED MANUSCRIPT panel, 100μm in GFAP staining panel. The data are presented as mean ± SD (6 rats per groups). ##: P < 0.01 compared with sham group. **: P < 0.01 compared with SCI
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group.
Figure 7. siRNA mediated Nrf2 knockout in the spinal cord tissue of rats. Intrathecal injection of Nrf2-siRNA significantly inhibited Nrf2 mRNA (A) and
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protein (B) expression in the spinal cord tissue of rats. The data are presented as mean
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± SD (6 rats per groups). ##: P < 0.01 compared with SCI+allicin 50 mg/kg group.
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Figure 8. Blockage of Nrf2 inhibits the protective effect of allicin. Intrathecal
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injection of Nrf2-siRNA decreased BBB scores compared with allicin therapy group
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(A). In addition, siRNA mediated Nrf2 gene knockdown markedly increased MDA level (B), decreased GSH level (C) and SOD activity (D) in the lesion site of spinal
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cord. The data are presented as mean ± SD (6 rats per groups). ##: P < 0.01 compared with sham group. **: P < 0.01 compared with SCI group. with allicin 50 mg/kg therapy group.
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S0024-3205(15)30065-5 doi: 10.1016/j.lfs.2015.11.001 LFS 14548
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
3 June 2015 12 October 2015 2 November 2015
Please cite this article as: Lv Runxiao, Mao Ningfang, Wu Jinhui, Lu Chunwen, Ding Muchen, Gu Xiaochuan, Wu Yungang, Shi Zhicai, Neuroprotective effect of allicin in a rat model of acute spinal cord injury, Life Sciences (2015), doi: 10.1016/j.lfs.2015.11.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Neuroprotective effect of allicin in a rat model of acute spinal cord injury
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Runxiao Lv1, Ningfang Mao2, Jinhui Wu1, Chunwen Lu1, Muchen Ding1, Xiaochuan
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Unit of Graduate Students, Changhai Hospital of Second Military Medical University,
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Shanghai 200433, People’s Republic of China
Department of Spine Surgery, Changhai Hospital of Second Military Medical
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2
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Gu1, Yungang Wu1, Zhicai Shi2, *
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University, Shanghai 200433, People’s Republic of China
*
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Corresponding author: Dr. Zhicai Shi, Email: [email protected]. Department of
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Spine Surgery, Changhai Hospital of Second Military Medical University, 168
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Changhai Road, Shanghai 200433, People’s Republic of China
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ACCEPTED MANUSCRIPT Abstract Aims: This study aims to investigate the effect of allicin on motor functions and
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histopathologic changes after spinal cord injury and the mechanism underlying its
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neuroprotective effects.
Main methods: The motor function of rats was evaluated with the Basso, Beattie, and Bresna test. Histopathologic changes were evaluated by hematoxylin and eosin and
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Nissl staining. Spinal cord oxidative stress markers were determined by measuring
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glutathione and malondialdehyde content and superoxide dismutase activity using commercial kits. Inflammatory factors were determined by measuring tumor necrosis
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factor-α, interleukin-1β and interleukin-6 using ELISA assay. Apoptosis was
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examined using TUNEL staining. The effect of allicin on Nrf2 protein levels and
analysis.
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localization was assessed using immunofluorescence staining and western blotting
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Key findings: Results demonstrated that allicin accelerated the motor functional recovery and protected neuron damage against spinal cord injury (SCI). SCI-induced oxidative stress, inflammatory response and cell apoptosis in the spinal cord were also prevented by allicin. In addition, we observed that SCI increased Nrf2 nuclear expression, and allicin treatment further increased Nrf2 nuclear translocation in neurons and astrocytes. siRNA-mediated Nrf2 gene knockdown completely blocked the effect of allicin on spinal cord tissue. Significance: Our finding suggests that allicin promotes the recovery of motor function after SCI in rats, and this effect may be related to its anti-oxidant, 2
ACCEPTED MANUSCRIPT anti-inflammatory and anti-apoptotic effects. Allicin mediated Nrf2 nuclear
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translocation may be involved in the protective effect as well.
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Keywords: spinal cord injury, allicin, apoptosis, inflammation, oxidative stress, Nrf2
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ACCEPTED MANUSCRIPT 1. Introduction Acute traumatic spinal cord injury (SCI) usually results a catastrophic neural
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dysfunction which negatively affects the quality of life of patients. The biphasic injury
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process in SCI includes primary and secondary injury mechanisms. The primary injury is caused by the initial physical impact, which is characterized by acute bleeding and ischemia. Following the primary insult, several pathways trigger the
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secondary injury, causing reactive glial changes, neuronal inflammation, oxidative
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stress, neuronal degeneration and apoptosis [1-4]. Even worse, the spontaneous anatomical and functional recovery will be prevented by these subsequent
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pathological events. Unfortunately, primary injury caused neuronal loss cannot be
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restored. Thus, most of the attention has been paid on the development of therapeutic
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strategy for the secondary injury. However, despite numerous promising experimental studies have been reported, effective treatment that can overcome secondary damage
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after SCI is lacking.
The nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is a classical signal that responsible for the cellular redox homeostasis. Activating this pathway is one of the main defense mechanisms against oxidative stress [5-7] and has been reported to be neuroprotective after SCI [8]. In SCI rats, Nrf2 levels were peaked at 30 min after SCI in cytoplasmic fractions and remained elevated for 3 days, and levels in nuclear fractions elevated at 30 min and peaked at 6 h following SCI. In addition, pharmacological activation of Nrf2/ARE pathway may contribute to the locomotion recovery and inhibit inflammatory 4
ACCEPTED MANUSCRIPT response [8, 9]. Allicin (diallyl thiosulfinate), is a small molecule extracted from the garlic. It is
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known as one of the most biologically active compounds in garlic, which is
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responsible for most of the functions of garlic [10]. Numerous studies have demonstrated the pharmacological effect of allicin, including anti-inflammatory, antimicrobial, antifungal, antiparasitic, antihypertensive, anti-diabetic and anti-tumor
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activities [11, 12]. Recent in vitro and in vivo studies reported that allicin could
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prevent traumatic neuronal injury in rat cortical neurons and traumatic brain injury rats by attenuating oxidative stress, inhibiting inflammatory response and neuron
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apoptosis [13, 14]. In addition, allicin has been found to have neuroprotective effect
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against spinal cord ischemia/reperfusion injury in rabbits [15]. However, its effect on
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acute traumatic spinal cord injury has not been investigated. In addition, Nrf2 activation has been approved to be beneficial in the recovery of SCI [16, 17]. In a
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previous study, allicin has been found to active Nrf2 signaling pathway in cardiac hypertrophy rats [18]. Therefore, we hypothesize the allicin would have neuroprotective effect via Nrf2 activation and aim to prove it in this study.
2. Material and Methods 2.1. Establishment of animal models of spinal cord injury A total of 90 female Sprague-Dawley rats weighing 200–250 g were purchased from Shanghai SLAC Laboratory Animal Co (Shanghai, China). All the animal experiments were performed in accordance with the guidelines of the Ethical 5
ACCEPTED MANUSCRIPT Committee of Experimental Animals of Second Military Medical University. After being acclimatized for 2 weeks, rats were randomly divided into the following groups
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(n=18): (1) sham; (2) SCI; (3) SCI+allicin 2mg/kg (allicin L); (4) SCI+allicin
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10mg/kg (allicin M); (5) SCI+allicin 50mg/kg (allicin H). All the rats were intraperitoneally anesthetized with 30 mg/kg pentobarbital sodium. The lower back was shaved and sterilized. A median incision was made on the back taking T8–9
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spinous process as a center to expose T7–10 spinous processes and the lamina and a
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laminectomy was performed at the T8 level. Rats in the sham group received only T8 laminectomy. Rats in the SCI groups received an injury in accordance with Allen's
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method. Briefly, a self-made 10 g rod was dropped vertically from a height of 50 mm
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on the exposed spinal cord. The rod was rest on the injury site for 3 min. The wound
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was then washed and the tissue was sutured. NaCl (5 mL of 0.9%) was injected intraperitoneally immediately after surgery, and penicillin (200,000 units/day,
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intramuscular injection) was used to protect from postoperative infection. Urinary bladder was manually emptied twice daily to assist in urination until the micturition function returned to normal. Rats in allicin groups received an intraperitoneally injection of allicin (MB5783, Meilun, Dalian, China) one hour before the surgery and once daily for 21 days. Rats in the sham and SCI groups received 0.9% NaCl daily.
2.2. siRNA design and delivery siRNAs encoding Nrf2 were purchased from GenePharma (Shanghai, China). Sequence
of
the
siRNA
used
in
the 6
present
study
is
as
followed:
ACCEPTED MANUSCRIPT CCGGAGAAAUUCCUCCCAAUTT. The negative scrambled sequence is as follows: UUCUCCGAACGUGUCACGUTT. After surgery, a hole was created at the exposed
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dura using a hypodermic syringe. An intrathecal catheter filled with sterile PBS was
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inserted 7 mm cephalad into the dura. The catheter exposed epidural was fixed tightly on the surrounding soft tissue. The end of the catheter was fixed on the skin and was sealed sterilely. Rats in siRNA treated groups received daily 5 μg/5 μl of Nrf2-siRNA
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or scramble siRNA through the intrathecal catheter for 3 days successively. The
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mRNA and protein expression of Nrf2 in the spinal cord was measured by real-time
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PCR and western blot, respectively.
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2.3.Evaluation of motor function
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Six rats in each group were randomly selected to perform motor function test at day 1, 4, 7, 14 and 21 after the surgery. Motor function was assessed using the Basso, Beattie
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and Bresnahan (BBB) locomotor rating scale [19]. The range of BBB locomotor scale scores was between 0 and 21. Twenty-one points refer to normal motor capacity and lower scores indicate impairment in motor capacity. Each rat was evaluated three times by three observers who were blinded to the treatment group, and the mean of the three measurements was taken.
2.4.Histological examinations After the 21-day treatment, six rats in each group were deeply anesthetized and perfused with 4% paraformaldehyde via the left ventricle for pre-fix. A 1.5 cm spinal 7
ACCEPTED MANUSCRIPT cord tissue surrounding the damage site was collected and post-fixed in 4% paraformaldehyde overnight. Fixed tissues were dehydrated using a series of ethanol,
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embedded in paraffin, and serially sectioned into 4 μm thick coronal slices. To
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perform staining, sections were deparaffinized in xylene and hydrated using a series of ethanol. Three sections of each group were used for hematoxylin and eosin (HE) (Solarbio Science & Technology, Beijing, China) staining. Three sections were
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stained with thionine (Solarbio). Five fields within each slide were randomly selected
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for neuronal counts. Sections were viewed under an optical microscope (DP73; Olympus, Tokyo, Japan). The cavity area was measured on sections stained with HE
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using IMAGE J software (National Institutes of Health, Bethesda, MD, USA).
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2.5.Tissue preparation and protein quantification After the 21-day treatment, the remained 6 rats in each group were deeply
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anesthetized and spinal cord tissue surrounding the damage site was immediately collected. Tissues were homogenized by a homogenizer in cooled RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) on ice. Homogenates were then centrifuged at 15,000 g for 10 min at 4°C. Nuclear and cytosolic proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) following the manufacturer’s instruction. The protein concentrations of the supernatants were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime).
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ACCEPTED MANUSCRIPT 2.6.Biochemical determination Spinal cord tissues were homogenized in cooled PBS and repeated freezing in liquid
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nitrogen and thawing for three times. Homogenates were then centrifuged at 15,000 g
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for 10 min at 4°C. Supernatants were used for the measurement of MDA and GSH levels and determination of SOD activity according to the protocols in the commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing,
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China).
2.7.Enzyme-linked immunosorbent assay (ELISA)
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Spinal cord protein supernatants were prepared in RIPA as described below. The
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determination of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 were
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performed using commercially available ELISA kits special for rats (USCN Life Science, Wuhan, China) according to the manufacturer’s instructions. Concentrations
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are given in pg/mg protein.
2.8.Real-time quantitative PCR Total RNA from tissue specimens in each group were purified using an RNA simple Total RNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's protocol. Oligonucleotide primer and super Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV) (BioTeke, Beijing, China) were used to make cDNA in a 20-μL reaction mixture. Quantitative real-time PCR was performed on 1-μL cDNA using 10-μL SYBR-Green Master Mix (Tiangen Biotechnology Co., Ltd., Beijing, 9
ACCEPTED MANUSCRIPT China), 1-μL cDNA and 10 μM of forward and reverse primers on an Exicycler™ 96 real-time quantitative thermal block (Bioneer, Daejeon, Korea) in a 20-μL reaction
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CTGGAATGGAAGGAGATGCC-3’, reverse: 5’-
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mixture. The sequences of primers are as follows: HO-1, forward: 5’-
TCAGAACAGCCGCCTCTACCG-3’, and β-actin, forward: 5’GGAGATTACTGCCCTGGCTCCTAGC-3’, reverse: 5’-
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GGCCGGACTCATCGTACTCCTGCTT-3’ (Sangon Biotech, Shanghai, China). The
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reaction was performed in an Exicycler™ 96 real-time quantitative thermal block
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(Bioneer, Taejon, Korea). Data were analyzed by using the 2−ΔΔCt method.
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2.9.Double immunofluorescence staining
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Dewaxed sections were incubated in Tris-Buffered saline (PBS) containing 0.1% Triton X-100 for 30 min and blocked with goat serum (Beyotime) for 30 min at room
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temperature. After being washed with PBS, sections were incubated with a mouse monoclonal anti-NeuN antibody (1:100, ab104224, Abcam, Cambridge, MA, USA) or a mouse monoclonal anti-GFAP antibody (1:50, sc-33673, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and a rabbit polyclonal anti-Nrf2 antibody (1:100, bs-1074R, Bioss, Beijing, China) overnight at 4°C. Sections were washed and then incubated in FITC labeled goat anti-mouse secondary antibody (A5608, 1:200, Beyotime) and Cy3 labeled goat anti-rabbit secondary antibody (A0516, 1:200, Beyotime), each for 1 hour at 37 °C in darkness. Sections were mounted in aqueous mounting medium (Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China). Fluorescent 10
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Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling
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(TUNEL)
Staining was performed using a TUNEL apoptosis detection kit (Wuhan Boster Biological Engineering Co., Ltd., Wuhan, China), according to the manufacturer's
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instruction. Sections were counterstained with haematoxylin (Solarbio) and mounted
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using Permount. Apoptotic cells that appeared brown under a microscope (DP73;
Western blot analysis
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2.11.
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Olympus) were counted in five fields of view per section, at 400× magnification.
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Equal amounts of protein were heat-denatured and loaded on a 12% sodium dodecyl sulfate-polyacrylamide gel to perform electrophoresis. Separated proteins were then
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transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using a wet transfer method. The membrane was blocked for 1 hour at room temperature with PBS containing 5% skimmed milk and 0.1% Tween-20, washed with 1 × Tris Buffered Saline, with Tween-20 (TBST) for three times and then incubated overnight at 4°C with primary antibodies specific for β-actin (1:1000, sc-47778, mouse monoclonal, Santa Cruz Biotechnology), Bcl-2 (1:400, BA0412, rabbit polyclonal, Boster, Wuhan, China), Bax (1:400, rabbit polyclonal, BA0315, Boster), cleaved-caspase 3 (1:500, bs-0081R, rabbit polyclonal, Bioss), Nrf2 (1:500, bs-1074R, rabbit polyclonal, Bioss), HO-1 (1:200, sc-10789, rabbit polyclonal, Santa 11
ACCEPTED MANUSCRIPT Cruz Biotechnology), and Histone H3 (1:1000, bs-17422R, rabbit polyclonal, Bioss) in the blocking buffer. The membrane was subsequently incubated with HRP
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conjugated secondary antibodies (1:5000; Beyotime), then visualized using an
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enhanced chemiluminescence system (7 Sea Pharmtech, Shanghai, China) and exposed on Fuji Rx 100 X-ray film (Fuji Photo Film, Tokyo, Japan). Bands were analyzed using Gel-Pro-Analyzer (Media Cybernetics, Bethesda, MD, USA). β-actin
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2.12.
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was taken as an loading control.
Statistical analysis
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All values are presented as the mean ± SD. Statistical analysis was performed using
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SPSS version 19.0 software (IBM, New York, NY, USA). The BBB scores were
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analyzed using Kruskal-Wallis test. Other data were analyzed using one-way analysis of variance and the least significant difference test. P value less than 0.05 was
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considered statistical significance.
3. Results
3.1. BBB test Rats were subjected to BBB test to evaluate the effect of allicin on functional improvements at 1, 4, 7, 14 and 21 days after surgery (Figure 1). The BBB score of rats in the sham group was 21, which indicated a normal motor function. One day after surgery, the motor function of rats was obviously impaired, represented by the dramatically decreased BBB scores. However, 14 days after allicin treatment, the 12
ACCEPTED MANUSCRIPT BBB score of rats in allicin 50 mg/kg group were significantly higher than that in the SCI group (P<0.05). At day 21, the score in allicin 50 mg/kg group reached higher
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(P<0.01 vs. SCI group) and allicin 10 mg/kg showed effective in improving motor
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functional recovery on SCI rats (P<0.05 vs. SCI group). There was not any significant difference between mean BBB scores of allicin 2 mg/kg and SCI groups (P>0.05).
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3.2. Effect of allicin on neuron damage in the spinal cords of SCI rats
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Neuron survival in the spinal cord was evaluated by HE and Nissl staining. Twenty-one days after surgery, marked necrosis, edema and loss of Nissl granules
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were observed in SCI groups (Figure 3). In the allicin 10 and 50 mg/kg treated groups,
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retained neurons with improved morphology and increased Nissl bodies were
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observed. These results indicate that allicin intervention may improve the histological
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changes of spinal cord and restore neuron function after SCI.
3.3.Effect of allicin on oxidative stress status in the spinal cords of SCI rats Oxidative stress status was examined by MDA level, GSH level and SOD activity assays. Following SCI, spinal cord tissue MDA level increased, and GSH level and SOD activity decreased significantly when the sham groups were compared with the SCI group (Figure 4, P < 0.01). Treatment with 10 and 50 mg/kg allicin significantly decreased the tissue MDA level and increased the tissue GSH level and SOD activity compared with the SCI group (P < 0.01). There was no significant difference between the 2 mg/kg allicin treatment and SCI groups though 2 mg/kg also decreased the mean 13
ACCEPTED MANUSCRIPT of those indexes (P > 0.05).
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3.4.Effect of allicin on inflammatory responses in the spinal cords of SCI rats
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Typical cytokines associated with inflammation (TNF-α, IL-1β and IL-6) in the spinal cord of each group 21-day post-injury were detected using ELISA. Levels of all the three cytokines were dramatically increased in the SCI group (Figure 5, P < 0.01).
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Allicin dose-dependently decreased these inflammatory cytokines secretion. Levels of
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TNF-α and IL-6 could be significantly decreased by 10 and 50 mg/kg allicin, and the
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level of IL-1β could be significantly decreased by all the 3 dosages of allicin.
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3.5.Effect of allicin on apoptosis in the spinal cords of SCI rats
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The cell apoptosis in the spinal lesions were detected by TUNEL staining. Sham group showed no apoptosis positive cells (Figure 5A). The numbers of
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TUNEL-positive cells increased significantly 21-day post-injury (Figure 5B, P<0.01 vs. sham group). Allicin dose-dependently decreased TUNEL-positive cells in the spinal cord (Figure 5C-E). In addition, expressions of apoptosis-associated proteins Bcl-2, Bax and cleaved-caspase 3 were also detected by western blot. In consistent with the result of TUNEL staining, allicin dose-dependently upregulated the expression of Bcl-2 and downregulated the expression of Bax and cleaved-caspase 3 (Figure 5H-K). Both of 10 and 50 mg/kg allicin showed the significant anti-apoptotic effect, whereas 2 mg/kg allicin has not shown significant effect.
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ACCEPTED MANUSCRIPT 3.6.Effect of allicin on Nrf2 nuclear translocation in the spinal cords of SCI rats The expression and nuclear translocation were detected using immunofluorescence
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staining and western blot. As shown in Figure 6A, following the surgery, Nrf2 protein
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showed an obvious nuclear translocation in both of the neuron and astrocyte, which was consistent with the previous study [8]. In addition, after allicin treatment, nuclear expression of Nrf2 was increased in 10 and 50 mg/kg allicin groups. The results of
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western blot analysis confirmed the observation in immunofluorescence staining.
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Nuclear expression of Nrf2 and expression of HO-1 was markedly upregulated by SCI and further upregulated by allicin 10 and 50 mg/kg treatment (Figure 6C-E). These
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impaired spinal cord.
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results suggested that allicin may promote Nrf2 nuclear translocation in the traumatic
3.7.Blockage of Nrf2 inhibits the protective effect of allicin
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In order to further study the role of Nrf2 in the protective effect of allicin, the siRNA-mediated gene knockdown was used. As illustrated in Figure 7, Nrf2-siRNA acute intrathecal injection successfully inhibited expression of Nrf2 in spinal cord tissue. In addition, Nrf2-siRNA intrathecal injection completely blocked the effect of allicin on SCI rats. The BBB scores were significantly lower than that in the allicin therapy group (Figure 8A). The scramble siRNA injection has not affected the therapeutic effect of allicin. In addition, Nrf2-siRNA also blocked the antioxidant effect of allicin. The level of MDA was significantly increased (Figure 8B) and level of GSH and activity of SOD were decreased (Figure 8C and D). These data suggested 15
ACCEPTED MANUSCRIPT that allicin protected rats from SCI through Nrf2 signal activation.
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4. Discussion
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In the present study, we demonstrated that allicin could improve motor function recovery of SCI rats, dose-dependently decreased neuronal death, attenuate oxidative stress and inflammatory response, and inhibite cell apoptosis in the lesion site of
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spinal cord. In addition, allicin mediated activation of Nrf2 signal may be involved in
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the protective effect of allicin.
Allicin has been demonstrated to have neuroprotective effects in some disease
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including neurodegenerative diseases and neurotrauma. In the present study, the
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neuroprotective effects of allicin were evaluated on SCI rats, which had not been
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previously studied. In the histopathological examination, acute SCI caused large cavity, hemorrhage, edema, and necrosis in the spinal cord. Additionally, in the SCI
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group, neurons with normal shape and function in the spinal cord were significantly decreased in number. This neuronal damage was consistent with the impaired motor function. Spinal cords of rats in both 10 and 50 mg/kg allicin groups showed better histological results and a high number of normal neurons compared with the SCI group. These two dosages of allicin treatment also improved motor function recovery in SCI rats at 21-day post-surgery. These results indicate that allicin has neuroprotective effect on spinal cord of SCI rats, which may contribute to the recovery of motor function. Primary and secondary injuries of acute spinal cord injury lead to neuronal death 16
ACCEPTED MANUSCRIPT in spinal cord. Among the secondary injury factors, oxidative stress, inflammation and apoptosis are the most important and have been paid most attention [20-25].
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Oxidative stress following SCI produces reactive oxygen species (ROS) and initiates
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lipid peroxidation activity in the injured neural tissue [26]. ROS leads damages to lipids, proteins and nucleic acids, and subsequently causes cytotoxicity [27]. The central nervous system is highly vulnerable to oxidative injury because the neural
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tissues consist largely of lipids. Previous studies demonstrated that allicin decreased
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reactive oxygen species (ROS) generation, reduced lipid peroxidation and increased antioxidant enzyme activities in various tissues including liver, kidney and spinal cord
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[28, 29]. In the present study, allicin inhibited oxidative stress by decreasing MDA
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level and increasing GSH level and SOD activity in a dose dependent manner, which
of allicin.
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indicates that the anti-oxidative effect may be involved in the neuroprotective effect
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Inflammatory responses play an important role in the secondary injury after SCI [30]. Elevated expressions of inflammatory cytokines have been demonstrated at both the mRNA and protein levels in spinal cords of animal models of SCI [31-33]. These cytokines are involved in neuronal necrosis and apoptosis, leucocyte infiltration and activation of glial cells, and play an important role in neurodegeneration in SCI [34, 35]. In the present study, 21 days after SCI, levels of TNF-α, IL-1β and IL-6 were significantly increased in the spinal cord. Allicin treatment markedly reduced the production of these inflammatory factors in a dose-dependent manner. This result suggests that the anti-inflammatory effect may be involved in the neuroprotective 17
ACCEPTED MANUSCRIPT effect of allicin. Acute SCI caused apoptosis leads to death of neuronal cells in the spinal cord,
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especially neurons and oligodendrocytes. This will further destroy the axon-myelin
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structure and impair impulse conduction, resulting in neuronal dysfunction [36, 37]. Neuronal apoptosis initiates as early as 4 hours around the lesion site after SCI, and neuronal and oligodendroglial apoptosis may last for weeks [38-40]. According to the
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previous studies, allicin has a biphasic effect on cell apoptosis. It has been
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demonstrated to induce apoptosis in numerous kinds of cancer cells [41-44]. However, it has also been shown to have anti-apoptotic effect on other diseases [45, 46]. In the
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present study, allicin effectively reduced number of apoptosis cells in the injured
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spinal cord tissue, upregulated the expression of Bcl-2 and downregulated expressions
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of Bax and cleaved-caspase 3, which are responsible for the mitochondrial pathway apoptosis. This result indicates the anti-apoptotic effect of allicin may contribute to its
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neuroprotective effect in SCI rats. The activation of Nrf2/ARE signal is dependent
upon the nuclear translocation
of Nrf2, which subsequently induces the transcriptions of protective genes [47]. In agreement with this theory, our results showed that SCI induces increased expression of Nrf2 in the nuclear of neurons and astrocytes. Allicin treatment after SCI further enhanced expression of the Nrf2 nuclear translocation resulting in increased expression of the HO-1. In response to oxidative stress, Nrf2 induces expressions of a number of antioxidant genes and other cytoprotective phase II detoxifying enzymes, such as 18
ACCEPTED MANUSCRIPT HO-1, SOD, glutathione peroxidase (GPx) and glutathione-S-transferases. Numerous studies have shown that Nrf2 knockout mice underwent highly susceptible to
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cytotoxic compounds compared to wild-type mice due to the decreased levels of
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antioxidant proteins and phase II detoxification enzymes [48-51]. In addition to anti-oxidant effect, overexpression of Nrf2 is also considered to protect cells from apoptosis and inflammatory response [52-54]. Our observation showed that the
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neuroprotective effect of allicin on SCI rats is Nrf2 activation-dependent, siRNA
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mediated Nrf2 gene knockdown completely blocked the effect of allicin, which indicated that allicin protected spinal cord tissue from traumatic injury through Nrf2
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activation.
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In this study, all the experiments were performed on female rats. It is well known
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that the menstrual cycle may affect some biological processes like inflammatory cytokines production, and the hormone may even have therapeutic effect on various
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disorders including SCI [55]. Although the menstrual cycle of animals has not been synchronized in this study, we found the data from each animal did not fluctuate much and the dose-dependent effect of allicin is remarkably. Thus we believe that the effect of menstrual cycle on our study is small and the neuroprotective effect seen in the SCI rats was come from allicin. 5. Conclusion In conclusion, the present study reveals that allicin exhibits neuroprotective effects through anti-apoptotic, anti-inflammatory, and anti oxidant effects on the injury spinal cord of rats. This effect is Nrf2 activation-dependent. This study 19
ACCEPTED MANUSCRIPT provides a novel neuroprotective drug candidate for SCI therapy.
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Conflict of interest statement
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The authors declare that they have no conflicts of interest concerning this article.
References
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[1] Block, Zecca, Hong, Microglia-mediated neurotoxicity: uncovering the molecular
MA
mechanisms, Nat Rev Neurosci 8 (2007) 57-69.
[2] Fleming, Norenberg, Ramsay, Dekaban, Marcillo, Saenz, et al., The cellular
D
inflammatory response in human spinal cords after injury, Brain 129 (2006)
TE
3249-3269.
89-105.
CE P
[3] Graeber, Streit, Microglia: biology and pathology, Acta Neuropathol 119 (2010)
AC
[4] Yiu, He, Glial inhibition of CNS axon regeneration, Nat Rev Neurosci 7 (2006) 617-627.
[5] McWalter, Higgins, McLellan, Henderson, Song, Thornalley, et al., Transcription factor Nrf2 is essential for induction of NAD(P)H:quinone oxidoreductase 1, glutathione S-transferases, and glutamate cysteine ligase by broccoli seeds and isothiocyanates, J Nutr 134 (2004) 3499S-3506S. [6] Alam, Stewart, Touchard, Boinapally, Choi, Cook, Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene, J Biol Chem 274 (1999) 26071-26078. 20
ACCEPTED MANUSCRIPT [7] Chanas, Jiang, McMahon, McWalter, McLellan, Elcombe, et al., Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression
IP
T
of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes
SC R
in the livers of male and female mice, Biochem J 365 (2002) 405-416. [8] Wang, de Rivero Vaccari, Wang, Diaz, German, Marcillo, et al., Activation of the nuclear factor E2-related factor 2/antioxidant response element pathway is
NU
neuroprotective after spinal cord injury, J Neurotrauma 29 (2012) 936-945.
MA
[9] Jin, Wang, Zhu, Yuan, Ni, Jiang, et al., Anti-inflammatory effects of curcumin in experimental spinal cord injury in rats, Inflamm Res 63 (2014) 381-387.
D
[10] Lawson, Gardner, Composition, stability, and bioavailability of garlic products
TE
used in a clinical trial, J Agric Food Chem 53 (2005) 6254-6261.
CE P
[11] Hunter, Caira, Stellenboom, Thiolsulfinate allicin from garlic: inspiration for a new antimicrobial agent, Ann N Y Acad Sci 1056 (2005) 234-241.
AC
[12] Bayan, Koulivand, Gorji, Garlic: a review of potential therapeutic effects, Avicenna J Phytomed 4 (2014) 1-14. [13] Zhou, Li, Han, Gao, He, Li, et al., Allicin protects rat cortical neurons against mechanical trauma injury by regulating nitric oxide synthase pathways, Brain Res Bull 100 (2014) 14-21. [14] Chen, Qi, Feng, Wang, Bao, Wang, et al., Neuroprotective effect of allicin against traumatic brain injury via Akt/endothelial nitric oxide synthase pathway-mediated anti-inflammatory and anti-oxidative activities, Neurochem Int 68 (2014) 28-37. [15] Zhu, Chen, Guan, Liu, Liu, Neuroprotective effects of allicin on spinal cord 21
ACCEPTED MANUSCRIPT ischemia-reperfusion injury via improvement of mitochondrial function in rabbits, Neurochem Int 61 (2012) 640-648.
IP
T
[16] Wang, Shen, Xie, Ling, Lu, Neuroprotective effect of ginseng against spinal cord
SC R
injury induced oxidative stress and inflammatory responses, Int J Clin Exp Med 8 (2015) 3514-3521.
[17] Jin, Ni, Hou, Ming, Wang, Yuan, et al., Tert-butylhydroquinone protects the spinal
NU
cord against inflammatory response produced by spinal cord injury, Ann Clin Lab Sci
MA
44 (2014) 151-157.
[18] Li, Li, Xiang, Hu, Lu, Tian, et al., Allicin ameliorates cardiac hypertrophy and
TE
Ther 26 (2012) 457-465.
D
fibrosis through enhancing of Nrf2 antioxidant signaling pathways, Cardiovasc Drugs
CE P
[19] Basso, Beattie, Bresnahan, A sensitive and reliable locomotor rating scale for open field testing in rats, J Neurotrauma 12 (1995) 1-21. Gul,
Bektas,
Hanci,
Acikgoz,
Effects
of
dexmedetomidine
or
AC
[20] Can,
methylprednisolone on inflammatory responses in spinal cord injury, Acta Anaesthesiol Scand 53 (2009) 1068-1072. [21] Beck, Nguyen, Galvan, Salazar, Woodruff, Anderson, Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment, Brain 133 (2010) 433-447. [22] Nakajima, Osuka, Seki, Gupta, Hara, Takayasu, et al., Taurine reduces inflammatory responses after spinal cord injury, J Neurotrauma 27 (2010) 403-410. 22
ACCEPTED MANUSCRIPT [23] Dinc, Iplikcioglu, Atabey, Eroglu, Topuz, Ipcioglu, et al., Comparison of deferoxamine and methylprednisolone: protective effect of pharmacological agents on
IP
T
lipid peroxidation in spinal cord injury in rats, Spine (Phila Pa 1976) 38 (2013)
SC R
E1649-1655.
[24] Vaziri, Lee, Lin, Lin, Sindhu, NAD(P)H oxidase, superoxide dismutase, catalase,
injury, Brain Res 995 (2004) 76-83.
NU
glutathione peroxidase and nitric oxide synthase expression in subacute spinal cord
Prog Brain Res 137 (2002) 37-47.
MA
[25] Beattie, Hermann, Rogers, Bresnahan, Cell death in models of spinal cord injury,
D
[26] Torres, Salgado-Ceballos, Torres, Orozco-Suarez, Diaz-Ruiz, Martinez, et al.,
TE
Early metabolic reactivation versus antioxidant therapy after a traumatic spinal cord
CE P
injury in adult rats, Neuropathology 30 (2010) 36-43. [27] Dawson, Dawson, Snyder, Molecular mechanisms of nitric oxide actions in the
AC
brain, Ann N Y Acad Sci 738 (1994) 76-85. [28] Abdel-Daim, Abdelkhalek, Hassan, Antagonistic activity of dietary allicin against deltamethrin-induced oxidative damage in freshwater Nile tilapia; Oreochromis niloticus, Ecotoxicol Environ Saf 111 (2015) 146-152. [29] Liu, Ren, Wang, Yao, He, Allicin protects spinal cord neurons from glutamate-induced oxidative stress through regulating the heat shock protein 70/inducible nitric oxide synthase pathway, Food Funct 6 (2015) 321-330. [30] Hayashi, Ueyama, Nemoto, Tamaki, Senba, Sequential mRNA expression for immediate early genes, cytokines, and neurotrophins in spinal cord injury, J 23
ACCEPTED MANUSCRIPT Neurotrauma 17 (2000) 203-218. [31] Wu, Yang, Mei, Yu, Selective inhibition of STAT1 reduces spinal cord injury in
IP
T
mice, Neurosci Lett 580 (2014) 7-11.
SC R
[32] Guo, Li, Shi, Wang, Yuan, Shi, et al., Genetic ablation of receptor for advanced glycation end products promotes functional recovery in mouse model of spinal cord injury, Mol Cell Biochem 390 (2014) 215-223.
NU
[33] Jiang, Tu, Zou, Hu, Wang, Li, et al., Neuroprotective effects of different
MA
modalities of acupuncture on traumatic spinal cord injury in rats, Evid Based Complement Alternat Med 2014 (2014) 431580.
D
[34] Lee, Yune, Baik, Shin, Du, Rhim, et al., Role of tumor necrosis factor-alpha in
TE
neuronal and glial apoptosis after spinal cord injury, Exp Neurol 166 (2000) 190-195.
CE P
[35] Hurst, Wilkinson, McLoughlin, Jones, Horiuchi, Yamamoto, et al., Il-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment
AC
seen during acute inflammation, Immunity 14 (2001) 705-714. [36] Crowe, Bresnahan, Shuman, Masters, Beattie, Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys, Nat Med 3 (1997) 73-76. [37] Pannu, Barbosa, Singh, Singh, Attenuation of acute inflammatory response by atorvastatin after spinal cord injury in rats, J Neurosci Res 79 (2005) 340-350. [38] Liu, Xu, Hu, Du, Zhang, McDonald, et al., Neuronal and glial apoptosis after traumatic spinal cord injury, J Neurosci 17 (1997) 5395-5406. [39] Casha, Yu, Fehlings, Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat, 24
ACCEPTED MANUSCRIPT Neuroscience 103 (2001) 203-218. [40] Shuman, Bresnahan, Beattie, Apoptosis of microglia and oligodendrocytes after
IP
T
spinal cord contusion in rats, J Neurosci Res 50 (1997) 798-808.
gastric
carcinoma
cell
line
through
SC R
[41] Zhang, Zhu, Duan, Feng, He, Allicin induces apoptosis of the MGC-803 human the
p38
mitogen-activated
protein
kinase/caspase-3 signaling pathway, Mol Med Rep 11 (2015) 2755-2760.
NU
[42] Chu, Ho, Chung, Raghu, Lo, Sheen, Allicin induces anti-human liver cancer cells
MA
through the p53 gene modulating apoptosis and autophagy, J Agric Food Chem 61 (2013) 9839-9848.
D
[43] Xu, Yu, Zhai, Zhang, Shen, Bai, et al., Role of JNK Activation and Mitochondrial
TE
Bax Translocation in Allicin-Induced Apoptosis in Human Ovarian Cancer SKOV3
CE P
Cells, Evid Based Complement Alternat Med 2014 (2014) 378684. [44] Cha, Choi, Cha, Choi, Cho, Allicin inhibits cell growth and induces apoptosis in
AC
U87MG human glioblastoma cells through an ERK-dependent pathway, Oncol Rep 28 (2012) 41-48.
[45] Chen, Tang, Qian, Chen, Zhang, Wo, et al., Allicin prevents H(2)O(2)-induced apoptosis of HUVECs by inhibiting an oxidative stress pathway, BMC Complement Altern Med 14 (2014) 321. [46] Liu, Qi, Wang, Wu, Cao, Huang, et al., Allicin protects against myocardial apoptosis and fibrosis in streptozotocin-induced diabetic rats, Phytomedicine 19 (2012) 693-698. [47] Liu, Li, Bu, Li, Li, Sun, et al., The neuroprotective potential of phase II enzyme 25
ACCEPTED MANUSCRIPT inducer on motor neuron survival in traumatic spinal cord injury in vitro, Cell Mol Neurobiol 28 (2008) 769-779.
IP
T
[48] Mao, Wang, Qiao, Wang, Disruption of Nrf2 enhances the upregulation of nuclear
SC R
factor-kappaB activity, tumor necrosis factor-alpha, and matrix metalloproteinase-9 after spinal cord injury in mice, Mediators Inflamm 2010 (2010) 238321. [49] Ramos-Gomez, Kwak, Dolan, Itoh, Yamamoto, Talalay, et al., Sensitivity to
NU
carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in
MA
nrf2 transcription factor-deficient mice, Proc Natl Acad Sci U S A 98 (2001) 3410-3415.
D
[50] Aoki, Sato, Nishimura, Takahashi, Itoh, Yamamoto, Accelerated DNA adduct
TE
formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust, Toxicol
CE P
Appl Pharmacol 173 (2001) 154-160. [51] Enomoto, Itoh, Nagayoshi, Haruta, Kimura, O'Connor, et al., High sensitivity of
AC
Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes, Toxicol Sci 59 (2001) 169-177. [52] Kotlo, Yehiely, Efimova, Harasty, Hesabi, Shchors, et al., Nrf2 is an inhibitor of the Fas pathway as identified by Achilles' Heel Method, a new function-based approach to gene identification in human cells, Oncogene 22 (2003) 797-806. [53] Li, Khor, Xu, Shen, Jeong, Yu, et al., Activation of Nrf2-antioxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis, Biochem Pharmacol 76 (2008) 1485-1489. 26
ACCEPTED MANUSCRIPT [54] Peng, Geh, Chen, Meng, Fan, Sartor, et al., Inhibitor of kappaB kinase beta regulates redox homeostasis by controlling the constitutive levels of glutathione, Mol
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Pharmacol 77 (2010) 784-792.
SC R
[55] Cox, Varma, Barry, Vertegel, Banik, Nanoparticle Estrogen in Rat Spinal Cord Injury Elicits Rapid Anti-Inflammatory Effects in Plasma, Cerebrospinal Fluid, and (2015).
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D
MA
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Tissue, J Neurotrauma
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ACCEPTED MANUSCRIPT Figure legends Figure 1 Influence of administration of allicin on the motor function of rats in
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21-day period of study. Allicin (10 and 50 mg/kg) treatment improved motor
± SD (6 rats per groups).
##
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functional recovery in SCI rats at day 21 post-injury. The data are presented as mean : P < 0.01 compared with sham group. *: P < 0.05
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compared with SCI group. **: P < 0.01 compared with SCI group.
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Figure 2 Influence of administration of allicin on spinal cord neurons. Haematoxylin and eosin and (A-E) Nissl staining (F-K) was performed to evaluate the
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effect of allicin on neurons in the spinal cord. (A and F) sham group, (B and G) SCI
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group, (C and H), SCI+allicin 2 mg/kg group, (D and I) SCI+allicin 10 mg/kg group,
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(E and J) SCI+allicin 50 mg/kg group, and (K) quantification of number of Nissl bodies. Scale bar: 100 μm. The data are presented as mean ± SD (6 rats per groups). ##:
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P < 0.01 compared with sham group. **: P < 0.01 compared with SCI group.
Figure 3 Influence of administration of allicin on the status of oxidative stress in the spinal cord of SCI rats. (A) Malondialdehyde (MDA) level, (B) glutathione (GSH) and (C) superoxide dismutase (SOD) activity. Allicin dose-dependently attenuated oxidative stress in the spinal cord. The data are presented as mean ± SD (6 rats per groups). ##: P < 0.01 compared with sham group. **: P < 0.01 compared with SCI group.
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ACCEPTED MANUSCRIPT Figure 4 Influence of administration of allicin on the inflammatory response in the spinal cord of SCI rats. Allicin dose-dependently decreased tumor necrosis
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factor-α (TNF-α), interleukin (IL)-1β and IL-6 secretion in the spinal cord of SCI rats.
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The data are presented as mean ± SD (6 rats per groups). ##: P < 0.01 compared with sham group. **: P < 0.01 compared with SCI group.
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Figure 5 Influence of administration of allicin on cell apoptosis in the spinal cord
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of SCI rats. Effect of allicin on cell apoptosis was evaluated using TUNEL staining (A-F) and western blot (G-J). (A) sham group, (B) SCI group, (C), SCI+allicin 2
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mg/kg group, (D) SCI+allicin 10 mg/kg group, (E) SCI+allicin 50 mg/kg group, and
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(F) quantification of apoptosis cells. (G) Blots of apoptotic-associated proteins, (H-J)
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semi-quantifications of protein levels. Allicin dose-dependently attenuated cell apoptosis in the spinal cord of SCI rats. Scale bar: 50 μm. The data are presented as
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mean ± SD (6 rats per groups). ##: P < 0.01 compared with sham group. **: P < 0.01 compared with SCI group.
Figure 6 Influence of administration of allicin on Nrf2 nuclear translocation in the spinal cord of SCI rats. (A) Double immunofluorescence staining of Nrf2 and NeuN or GFAP. Nrf2 immunoreactivity increases after SCI and allicin administration in neurons and astrocytes of the spinal cord. (B) Protein expression of cytoplasmic Nrf2, (C) protein expression of nuclear Nrf2, (D) protein expression of HO-1, and (E) mRNA expression of HO-1 in the spinal cord. Scale bar: 50 μm in NeuN staining 29
ACCEPTED MANUSCRIPT panel, 100μm in GFAP staining panel. The data are presented as mean ± SD (6 rats per groups). ##: P < 0.01 compared with sham group. **: P < 0.01 compared with SCI
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group.
Figure 7. siRNA mediated Nrf2 knockout in the spinal cord tissue of rats. Intrathecal injection of Nrf2-siRNA significantly inhibited Nrf2 mRNA (A) and
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protein (B) expression in the spinal cord tissue of rats. The data are presented as mean
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± SD (6 rats per groups). ##: P < 0.01 compared with SCI+allicin 50 mg/kg group.
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Figure 8. Blockage of Nrf2 inhibits the protective effect of allicin. Intrathecal
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injection of Nrf2-siRNA decreased BBB scores compared with allicin therapy group
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(A). In addition, siRNA mediated Nrf2 gene knockdown markedly increased MDA level (B), decreased GSH level (C) and SOD activity (D) in the lesion site of spinal
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cord. The data are presented as mean ± SD (6 rats per groups). ##: P < 0.01 compared with sham group. **: P < 0.01 compared with SCI group. with allicin 50 mg/kg therapy group.
30
§§
: P < 0.01 compared
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Figure 1
31
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ACCEPTED MANUSCRIPT
Figure 2
32
SC R
IP
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ACCEPTED MANUSCRIPT
AC
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Figure 3
33
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
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Figure 4
34
AC
Figure 5
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IP
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ACCEPTED MANUSCRIPT
35
AC
CE P
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D
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Figure 6
36
SC R
IP
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AC
CE P
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Figure 7
37
AC
Figure 8
CE P
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SC R
IP
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ACCEPTED MANUSCRIPT
38