AMPK signaling pathway

Accepted Manuscript Resveratrol Protects Against Spinal Cord Injury by Activating Autophagy and Inhibiting Apoptosis Mediated by the SIRT1/AMPK Signaling Pathway Haosen Zhao, Shurui Chen, Kai Gao, Zipeng Zhou, Chen Wang, Zhaoliang Shen, Yue Guo, Zhuo Li, Zhanghui Wan, Chang Liu, Xifan Mei PII: DOI: Reference:

S0306-4522(17)30110-0 http://dx.doi.org/10.1016/j.neuroscience.2017.02.027 NSC 17616

To appear in:

Neuroscience

Received Date: Revised Date: Accepted Date:

15 June 2016 1 February 2017 14 February 2017

Please cite this article as: H. Zhao, S. Chen, K. Gao, Z. Zhou, C. Wang, Z. Shen, Y. Guo, Z. Li, Z. Wan, C. Liu, X. Mei, Resveratrol Protects Against Spinal Cord Injury by Activating Autophagy and Inhibiting Apoptosis Mediated by the SIRT1/AMPK Signaling Pathway, Neuroscience (2017), doi: http://dx.doi.org/10.1016/j.neuroscience. 2017.02.027

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.

Resveratrol Protects Against Spinal Cord Injury by Activating Autophagy and Inhibiting Apoptosis Mediated by the SIRT1/AMPK Signaling Pathway Haosen Zhaoa#, Shurui Chena#, Kai Gaob, Zipeng Zhoua, Chen Wanga, Zhaoliang Shenc, Yue Guoa, Zhuo Lic, Zhanghui Wana, Chang Liud, *, Xifan Meia, * a

Department of Orthopedic, First Affiliated Hospital of Jinzhou Medical

University, Jinzhou City, PR China b

Department of Orthopedics, Jining NO.1 People’s Hospital, Jining City,

PR China c

Department of Orthopedics, Second Hospital of Jinzhou, Jinzhou, PR

China d

Department of Endocrinology, First Affiliated Hospital of Jinzhou

Medical University, Jinzhou City, PR China * These authors contributed equally to this work and should be considered co-correspondence authors Prof. Chang Liu, E-mail address: [email protected] Prof. Xifan Mei, E-mail address: [email protected] # These authors contributed equally to this work and should be considered co-first authors: Haosen Zhao, E-mail address: [email protected] Shurui Chen, E-mail address: [email protected]

Abstract Spinal cord injury (SCI) is a devastating condition with few effective treatments. Resveratrol, a polyphenolic compound, has exhibited neuroprotective effects in many neurodegenerative diseases. However, the explicit effect and mechanism of resveratrol on SCI is still unclear. Adenosine 5’ monophosphate-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1)，the downstream protein, play key roles in metabolizing of energy, resisting of resistance, and cellular protein homeostasis. In this study, we determined the effects of resveratrol on SCI and their potential relationship with SIRT1/AMPK signaling pathway, autophagy and apoptosis. To determine the effect of resveratrol on SCI recovery, a spinal cord contusion model was employed. Rats received treatment with resveratrol or DMSO immediately following contusion. We determined that Basso, Beattie, and Bresnahan (BBB) scores were significantly higher for injured rats treated with resveratrol. Nissl and HE staining revealed that resveratrol treatment significantly reduced the loss of motor neurons and lesion size in the spinal cord of injured rats when compared to vehicle-treated animals. Spinal cord tissue was assessed by Western blot, reverse transcriotion-polymerase chain reaction (RT-PCR) and immunohistochemical analyses 7 days after injury for changes in expression of SIRT1/AMPK signaling pathway, autophagy and apoptosis proteins. Expression of SIRT1, p-AMPK, Beclin-1, LC3-B, and Bcl-2

was elevated in resveratrol-treated animals, whereas expression of p62, Cleaved Caspase-3, Caspase-9, and Bcl-2 associated X protein (Bax) was inhibited. Immunofluorescence analysis of primary neurons treated with resveratrol alone or in combination with Compound C (AMPK inhibitor) or EX527 (SIRT1 inhibitor) revealed that treatment with the inhibitors blocks the increase LC3-B expression in cells and increases the portion of TUNEL positive cells. Taken together, these results suggest that resveratrol exerts neuroprotective effects on SCI by regulating autophagy and apoptosis mediated by the SIRT1-AMPK signaling pathway. Introduction Spinal cord injury (SCI) consists of two injuries: primary injuries that occur at initial impact and secondary injuries that develop soon after the injury. Primary injuries include mechanical compression of the spine. The heterogeneity and complexity of the damage to the spinal tissue makes these injuries particularly difficult to treat. Secondary SCI effects such as post-traumatic inflammation, oxidative stress, motor neuron apoptosis and necrosis, and autophagy of local tissue cause additional damage to the initial injury resulting in a dynamic disease with ever changing debilitating consequences (Tator and Fehlings, 1991). Numerous studies have shown that secondary injury processes can be minimized by various measures (Toerge et al. 1978; Isaac and Pejic, 1995). Thus, treatment and prevention of secondary injury may improve treatment and recovery of

SCIs. Potential therapeutic targets for secondary SCI are autophagy and apoptosis mechanisms. One of the most devastating processes of secondary SCI is neuronal apoptosis (Lou et al. 1998). Motor neuron apoptosis is the main cause of dysfunction after SCI, as it is one of the primary obstacles for the locomotor functional recovery (Lu et al. 2000). Recent studies have found common molecular mechanisms between apoptosis and autophagy, which suggests an inverse relationship between autophagy and apoptosis mechanisms. Autophagy, the intracellular degradation of proteins and organelles, aims to produce energy for adaptation and survival under starvation (Apel et al. 2008). Mounting evidence indicates that autophagy may serve as a neuroprotective in neurodegenerative diseases. A study by Mizushima found that activation of autophagy results in tissue sparing following SCI in rats (Mizushima et al. 2008). Thus, activation of autophagy mechanisms may aid in the recovery and treatment of spinal tissue following SCI. A promising therapy that may utilize these mechanisms is resveratrol. Resveratrol is a natural polyphenolic compound that exhibits a multitude of beneficial health properties including anti-oxidant, anti-inflammatory, and anti-tumor effects (Kraft et al. 2009; Kalantari and Das, 2010; Fernández and Fraga, 2011; Feng et al. 2016). Resveratrol has been

shown to activate autophagy through upregulation of Sirtuin 1 (SIRT1) (Denu, 2012; Gut and Verdin, 2013). There is also evidence that resveratrol can function as a neuroprotective by regulating apoptosis (Lin et al. 2011; Bastianetto et al. 2014; Bellaver et al. 2014). A number of recent studies have also found that resveratrol may be effective in treating spinal cord injury (Liu et al. 2011; Kesherwani et al. 2013). Sirtuin 2 enzymes are a highly conserved NAD+-dependent histone deacetylase. Humans possess seven Sir2 homologous genes: SIRT1 to SIRT7, of which SIRT1 exhibits the highest homology. SIRT1 has become a hot topic in recent years (Berggren, 2006; Milner and Allison, 2011). Recent studies have shown that small molecule modulators of sirtuins exhibit promising therapeutic effects for the treatment of age-related diseases, inflammatory disorders, and cardiovascular and neurodegenerative diseases. Among them, SIRT1, as a downstream protein of SIRT1/AMPK signaling pathway, plays a key role in cell survival, energy metabolism, and aging (Sedding and Haendeler, 2007; Satoh and Imai, 2014). Zhang et al. (2016) indicate that the protective effects of SIRT1 may be related to the activation of autophagy, thereby inhibiting apoptosis. Adenosine 5’ monophosphate-activated protein kinase (AMPK) functions as a cellular energy detector (Xiao et al. 2011). Many studies prove the anti-aging and anti-senescence role of AMPK. The activation of

AMPK showed beneficial effects for stabilizing intracellular environment and preventing senescence (Apfeld et al. 2004; Stenesen et al. 2013; Ido et al. 2014). Studies show that the role of AMPK in aging prevention was generally because of the activation effects of Sirt1 and FoxO1 (Wang and Vanhoutte, 2011; Yun et al. 2014). The mutual regulations of the SIRT1 and AMPK signaling pathways have been observed in different tissues and cell types. AMPK signaling also activates autophagy (Salminen and Kai, 2012; Medina et al. 2015). Despite its therapeutic potential in treating spinal cord injuries, the mechanisms underlying resveratrol’s function in spinal cord injury are unclear. In the present study, we investigate the effect of resveratrol on functional recovery and tissue sparing using a rat spinal cord contusion model and identify a novel mechanism of action in SCI. We determined that resveratrol improves functional recovery, protects against motor neuron loss and lesion expansion following SCI. These improvements in recovery are accompanied by enhancement of the autophagy and inhibition of apoptosis regulated by SIRT1/AMPK signaling pathway. Keywords Spinal Cord Injury; Resveratrol; SIRT1/AMPK Signaling Pathway; Autophagy Experimental Procedures Chemical Reagents

Resveratrol (Dalian Meilun, China) was dissolved in dimethyl sulfoxide (DMSO) (Sigma, USA) and then diluted with saline to a final DMSO concentration of 2%. Animal Care Female Sprague–Dawley rats (weighing 220–240 g) were purchased from Vitalriver, Beijing and raised in the specific pathogen free laboratory animal center of Jinzhou Medical University with an alternating 12 h day and night cycle and constant environment of 25 ± 0.5 ℃. All experimental procedures complied with the Experimental Animal Ethics Committee of Jinzhou Medical University. Spinal Cord Injury Spinal cord contusion was conducted in accordance with Allen’s method (Allen, 1911). Animals were anesthetized by injection of chloral hydrate (3 mL/kg, i.p.). Following anesthesia, rats were placed in the prone position and the spinal cord was exposed by laminectomy at lamina T9-10 aseptically. A 2 mm diameter, 10 g impounder was dropped from a height of 30 mm on the surface of the T9-10 spinal cord, leading to spinal cord congestion. Animals in the sham group underwent laminectomy only. Bladder was expressed manually twice a day until bladder function was recovered. Resveratrol Treatment The rats were randomly divided into three groups: sham, vehicle (2%

DMSO) and resveratrol (100 mg/kg) (Bao et al. 2003). The vehicle or resveratrol was administered by intraperitoneal injection immediately after spinal cord injury. Behavioral Assessment The functional neurological deficits due to the SCI were assessed by behavioral analysis using the Basso, Beattie, and Bresnahan (BBB) open-field locomotor test (Basso et al. 1995). Double-blind assessment was performed before injury and at 0, 1, 3, 7, 14, 21, and 28 days post-injury. BBB scores range from 0 to 21 points. The 0 point indicates complete immobility, and 21 points indicates for normal function. The average scores were calculated in accordance with the grading standard in locomotion recovery after SCI. Primary neuronal culture Pregnant rats were euthanized and a cesarean section was performed 18 days into the pregnancy. The spinal cords of fetal rats were removed, well-minced and digested in papain (Solarbio, China). Cells were seeded at concentration of 1 × 10 6 cells/well in a 6-well plate for immunofluorescence and TUNEL staining analysis. Well plates were pretreated with poly-d-lysine (Sigma, USA) one day before seeding. The cells were coated with a mixture of Dulbecco's Modified Eagle Medium (DMEM, Hyclone, USA), 10% fetal bovine serum (Gibco, USA), and 0.5% antibiotics (penicillin/streptomycin, Gibco, USA) for 8 h. The

medium was then changed to a neurobasal media composed of B27 (1%, Gibco,

USA),

GlutaMAX

(0.25%,

Gibco,

USA),

and

penicillin/streptomycin (0.5%, Gibco, USA). The cells were cultured in an incubator at 37 °C and 5% CO2 for 7 to 10 days before use in follow-up experiments. Cell Treatment Five groups were set up, namely control group, lipopolysaccharides (LPS) group, resveratrol group, Compound C group and EX527 group. In LPS group, cells were treated with LPS (100 ng/mL, Sigma, USA) for 6 h. While in resveratrol group, Compound C group and EX527 group, cells were incubated with LPS and resveratrol (10 μM) together. Compound C (2 μM, Selleck, USA) and EX527 (2 μM, Selleck, USA) were added into Compound C group and EX527 group respectively. Tissue Preparation For detection of Western blot and reverse transcription-polymerase chain reaction (RT-PCR), the spinal cord was removed directly after euthanasia and cut from the epicenter at 7 day after spinal cord injury. Half was kept for the Western blot, while the other half was kept for RT-PCR. For staining detection, the rats were perfused with 0.9% saline and 4% paraformaldehyde (PFA). The spinal cord tissue was removed (the epicenter included) and the tissues were immersed in 4% PFA for 24 h. The immersion liquid was then replaced with sucrose solution (30%).

Approximately

5

μm

crosswise

sections

were

prepared

for

immunofluorescence staining, 20 μm crosswise sections for Nissl staining, and 20 μm portrait sections for HE staining using the cryostat microtome (Leica, Germany). Western Blot Tissue preparations were dissolved in RIPA lysis buffer (Beyotime, China). The final protein concentration (2 μg/μL) was quantified using the BCA kit (Enogene, China). Protein samples (40 μg) were separated by SDS-PAGE and transferred into PVDF membranes. After blocking in 5% skimmed milk powder dissolved in Tris-buffered Saline with Tween 20 (TBST) for 2 h at room temperature (RT), the membranes were incubated at 4 °C overnight in primary antibodies. Primary antibodies include anti-SIRT1 (1:1000, Cell Signalling, USA), anti-p-AMPK (1:1000, Cell Signaling, USA), anti-Cleaved Caspase-3 (1:1000, Novus, USA), anti-Caspase-9 (1:1000, Novus, USA), anti-Bcl-2 (1:1000, Abcam, USA), anti-Bax (1:1000, Santa Cruz, USA), anti-Beclin-1 (1:500, Novus, USA), anti-LC3-B (1:1000, Abcam, USA), anti-p62 (1:1000, Abcam, USA) and anti-β-actin (1:1000, Transgene, China). Membranes were washed with TBST thrice and incubated with secondary antibodies (1:10,000; Earthox, USA) at RT for 2 h. The membranes were developed using ECL kit (Beyotime, China) in ChemiDoc-It TS2 Imager (UVP). Relative optical density was performed using ImageJ 2X software.

RT-PCR Analysis The total RNA from spinal cord tissues prepared in advance was extracted using TRIZOL reagent (Invitrogen, USA). cDNA was synthesized from 1 μg of the total RNA using the TaKaRa RNA PCR™ kit (AMV) Ver. 3.0 (Takara, Japan). The mRNA expression of SIRT1, AMPK, Beclin-1, Bcl-2, Bax, and β-actin was inspected using cDNA as a template for amplification utilizing the following primers: SIRT1 (forward primer 5′-ATGATTGGCACCGATCCTCG-3′ and reverse primer 5′-ATTCCTGCAACCTGCTCCAA-3′); AMPK (forward primer 5′-GAAGATCGGACACTACGTGC-3′

and

5′-AGTCCACGGCAGACAGAATC-3′);

Beclin-1

5′-AAAGAGTGGAAGATGTCCGGC-3′

reverse

primer

(forward

primer

and

reverse

primer

5′-CAGCTGCTTCTCACCCTTGTA-3′);

Bcl-2

(forward

primer

5′-GGGGCTACGAGTGGGATACT-3′

and

reverse

primer

(forward

primer

reverse

primer

5′-GACGGTAGCGACGAGAGAAG-3′); 5′-TGGCGATGAACTGGACAACA-3′

Bax and

5′-TAGAAAAGGGCAACCACCCG-3′); and β-actin (forward primer 5′-ATATCGCTGCGCTCGTCG-3′

and

reverse

primer

5′-CAATGCCGTGTTCAATGGGG-3′). The cycling conditions were as follows: 5 min at 92 °C followed by 27 cycles of 30 s at 92 °C, 30 s at 50 °C and 30 s at 70 °C. Final extension was performed at 70 °C for 10 min.

Nissl Staining Immersed in the solution of ethanol and chloroform (1:1, v/v) overnight， 20 μm crosswise sections were dehydrated in a set of alcohol with decreasing concentration: 100% alcohol, 95% alcohol, and distilled water. Sections were stained in preheated 0.05% (w/v) cresyl violet solution for 20 s at 37 °C. The sections were then rinse in distilled water quickly and differentiated in HCl/95% alcohol (1:50) solution for 10 min. After dehydration in 100% alcohol and clearance using xylene, the sections were mounted with neutral gum. Hematoxylin and Eosin (HE) staining Following staining with hematoxylin for 30 s, 20 μm portrait sections were rinsed in distilled water quickly. The slides were differentiated in HCl/95% alcohol (1:50) solution for 6 s. After washing with distilled water for 1 h, the sections were restained with eosin. The sections were mounted with neutral gum after dehydration through increasing concentrations of ethanol, namely 75% alcohol, 95% alcohol, and 100% alcohol and clearance with xylene. Immunofluorescence Analysis The pre-treated primary neurons were fixed with 4% PFA at RT for 30 min. The 5 μm sections were air dried at RT for 2 h. Then, the cells and sections were blocked with normal goat serum blocking buffer (ZSGB-Bio, China) at 4 °C for 2 h. The cells and sections were incubated

with the primary antibodies overnight: anti-LC3-B (1:200, Abcam, USA) for the cells, anti-LC3-B (1:200, Abcam, USA) and anti-Cleaved Caspase-3 (1:200, Novus, USA) for the sections. The cells were then incubated in FITC goat anti-rabbit IgG (ZSGB-Bio, China) for 30 min at RT and followed by incubation with primary anti-NeuN (1:500, Abcam, USA) and Cy3 goat anti-mouse IgG (ZSGB-Bio, China) for 2 h at room temperature. The sections underwent a final incubation in FITC goat anti-rabbit IgG (ZSGB-Bio, China) and Cy3 goat anti-rabbit IgG (ZSGB-Bio, China) respectively after incubated with anti-LC3-B and anti-Cleaved Caspase-3. Parts of the anti-Cleaved Caspase-3 incubated sections were then incubated with anti-p62 (1:200, Abcam, USA) and FITC goat anti-mouse IgG (ZSGB-Bio, China) for 2 h at room temperature. Finally, the nucleus was redyed with DAPI solution (1:1000). All slides were mounted on a mounting medium and observed using a fluorescence microscope (Leica, Germany). TUNEL staining After washing with 1×phosphate buffer saline (PBS) twice and fixing with 4% PFA for 30 min at RT, the neurons were incubated with 1× equilibration buffer for 10 min at RT followed by the TUNEL reaction mixture (TUNEL Apo-Green Apoptosis Detection Kit, Biotool, USA) for 1 h at 37 °C. The cells were rinsed with 1×PBS twice, and the nucleus was counterstained with DAPI (1:1000) for 10 min. The cells were

observed using a fluorescence microscope (Leica, Germany). Statistical Analysis All data are presented as mean ± SD. Unpaired Student’s t-test and one-way ANOVA were used for comparison of two groups or multi-groups, respectively. The Mann–Whitney U test was used for the analysis of BBB scores. Statistical analyses were carried out using the SPSS 19.0. p < 0.05 was considered statistically significant. Results Resveratrol enhances motor function recovery following SCI in rats To determine the effect of resveratrol on locomotor function following SCI, spinal cord contusion was performed on female Sprague Dawley rats at T9-T10. Animals received resveratrol (100 mg/kg) or vehicle immediately after injury. Locomotor was assessed before and on various days following injury using the BBB locomotor test. Both vehicle and resveratrol treated rats demonstrated hindlimb paralysis immediately following injury. Rats treated with resveratrol scored significantly higher than rats receiving the vehicle on 14 and 28 days post-injury (Figure 1) (p < 0.05). Resveratrol increases the survival of motor neurons and reduces lesion size after SCI in rats To determine if resveratrol improves tissue sparing following SCI, the motor neurons in the anterior horn of the spinal cord from SCI rats

receiving resveratrol was examined. Tissue was collected 7 days after injury and underwent Nissl staining and HE staining. Motor neurons were counted following Nissl stain. Rats treated with resveratrol had significantly more motor neurons in the anterior horn than rats that received the vehicle (Figure 2A&C). Moreover, lesion size was significantly smaller in resveratrol treated rats compared to rats treated with vehicle only (Figure 2B&D). Resveratrol upregulates the SIRT1/AMPK signaling pathway and promotes autophagy after SCI in rats To determine if resveratrol promotes recovery following SCI by activating autophagy, we examined expression of autophagy in spinal cord tissue. We found that protein expression of SIRT1, p-AMPK, Beclin-1, and LC3-B was significantly higher 7 days post-injury in resveratrol-treated rats than in rats that received the vehicle or sham rats (Figures

3A-3E)

while

the

expression

of

p62

was

reduced

correspondingly in resveratrol-treated animals (Figure 3F). mRNA expression of SIRT1, AMPK, and Beclin-1 was significantly higher in resveratrol-treated rats than untreated rats as well (Figures 4A-4D). Immunofluorescence analysis also showed significant increase of LC3-B in the spinal cord of rats received resveratrol treatment (Figure 5A&B). Taken together, the results suggest that resveratrol regulates the SIRT1/AMPK signaling pathway and promotes autophagy in SCI rats.

Resveratrol inhibits apoptosis after SCI in rats To determine whether resveratrol reduces motor neuron loss and lesion size by inhibiting apoptosis, we examined expression of apoptosis proteins in the spinal cord of treated and untreated SCI rats. We determined that Cleaved Caspase-3, Caspase-9, and Bax was upregulated in rats that received the vehicle compared to sham rats, while Bcl-2 was markedly down-regulated. However, employment of resveratrol reduced Cleaved Caspase-3, Caspase-9, and Bax, and increased the expression levels of Bcl-2 significantly post-injury (Figures 6A-6E). Changes in mRNA expression of Bcl-2 and Bax reflected those determined by protein expression (Figures 7A-7C). Moreover, immunofluorescence analysis also showed that Cleaved Caspase-3 expression was significantly downregulated in the spinal cord of rats that received resveratrol (Figures 8A&B). These results reveal that resveratrol markedly suppresses apoptosis in SCI rats. Resveratrol upregulates autophagy and inhibits apoptosis after SCI in rats To determine the association of autophagy upregulation and block of apoptosis

by

resveratrol

treatment

after

SCI,

we

conducted

coimmunostaining of p62 and Cleaved Caspase-3 (Figures 9A). We determined that Cleaved Caspase-3 was upregulated in rats that received the vehicle compared to sham rats, while p62 was markedly

down-regulated. However, employment of resveratrol reduced Cleaved Caspase-3 and the expression levels of p62 significantly post-injury (Figures 9B&C). These results show the association of autophagy upregulation and block of apoptosis. Inhibition of SIRT1/AMPK signaling pathway decreases expression of autophagy and increases apoptosis expression Immunofluorescence analysis of primary neurons revealed that during treatment with Compound C or EX527, the proportion of LC3B-positive neurons decreased significantly (Figures 10A&B). The result indicates that the upregulation effect of resveratrol is related to the activation of SIRT1/AMPK pathway. In addition, the result of TUNEL staining shows significant increase of TUNEL-positive neurons in Compound C or EX527-treated group (Figures 11A&B). Taken together, these results suggest that resveratrol enhances autophagy and inhibits apoptosis by inducing the SIRT1/AMPK signaling pathway, and that there may be an inverse relationship between autophagy and apoptosis. Discussion The present study demonstrated that resveratrol promotes locomotor recovery and motor neuron survival, and reduces injury size in the spinal cord of SCI rats. Resveratrol also increased expression of SIRT1, p-AMPK, and Beclin-1, LC3-B, key proteins of autophagy and inhibited expression of apoptosis proteins.

Previous

studies

have

reported

that

resversatol

exerts

neuroprotective effects by regulating apoptosis (Lin et al. 2011; Bastianetto et al. 2014; Bellaver et al. 2014). Our findings are consistent with these studies. The resveratrol-treated SCI rats showed significant functional recovery, reduced loss of motor neurons, and reduction of the spinal cord lesion size. Expression of apoptosis was significantly suppressed on protein and mRNA level as shown by Western blot, RT-PCR, and immunofluorescence analyses. We also found that inhibition of SIRT1 or AMPK inhibited expression of

LC3-B and enhanced expression of apoptosis, which

suggests that resveratrol performed as a neuroprotective and promoted functional recovery through regulation of apoptosis and autophagy mediated by the SIRT1/AMPK signaling pathway. Autophagy occurs mainly for the turnover of damaged or long-lived proteins at low levels under normal circumstances. When the environment introduces new stresses, autophagy is activated to protect cells against these stresses. This mechanism is particularly important in fighting

against

multiple

diseases,

such

as

infections

and

neurodegenerative disorders (Galluzzi et al. 2008; Criollo et al. 2010). Although controversial, upregulation of autophagy after SCI is considered to play a neuroprotective role in recovery (Lipinski, et al. 2015). Previous studies have found that when autophagy is inhibited after

central nervous system injury, neuronal death increases. He et al. showed that the protective effect of autophagy after SCI is related to its role in stabilization of microtubules and promotion of axonal regeneration (He et al. 2016). More studies have shown that upregulation of autophagy after SCI can also reduce the incidence of apoptosis (Zhou, et al. 2016; Li, et al. 2016). Our findings are consistent with these findings to a certain extent. Autophagy and apoptosis exhibit crosstalks in a consistent degree, at numerous levels. For example As caspases are activated, Beclin-1, an essential protein for autophagy decreases and leads to the inhibited autophagic pathway (Maiuri et al. 2010). Some cellular stress sensing molecular mechanisms can induce not only autophagy but also apoptosis. Our results also indicated that the autophagy expression increased after the resveratrol treatment on SCI compared with the vehicle group, while apoptosis was inhibited, indicating that the neuroprotective and anti-apoptosis effects of resveratrol may be related with the activation of autophagy. We determined that AMPK and SIRT1 are specifically elevated by resveratrol. AMPK is a fundamental regulator of energy metabolism, stress tolerance, cellular protein homeostasis and senescence prevention (Salminen et al. 2016). SIRT1, a member of the highly conserved family of NAD+-dependent protein lysine-modifying enzymes, is another key regulator of cellular metabolic homeostasis. Although existing as a

downstream of AMPK, studies demonstrate that SIRT1 and AMPK exhibit a consistent degree of crosstalk. As sensors of cellular energy homeostasis and regulator of stress resistance, both SIRT1 and AMPK mediate autophagy. Resveratrol is generally regarded as an agonist of SIRT1. However, studies show that resveratrol could also activate AMPK and in hepatocellular carcinoma cells, resveratrol could activate AMPK signaling in SIRT1-dependent or not ways (Hou et al. 2008; Shin et al. 2009). Our results are consistent with these studies. In rats after SCI, treatment with resveratrol significantly upregulated the expression of SIRT1 and AMPK in Western blot and RT-PCR detection. However, in neurons treated with Compound C or EX527, the inhibitor of AMPK and SIRT1, respectively, the LC3-B level of immunofluorescence analysis was suppressed significantly, in comparison with resveratrol treatment. Correspondingly, the proportion of neurons shown TUNEL positive increased significantly. These results indicated that the anti-apoptosis effects of resveratrol were related to the activation of autophagy by the up-regulation of SIRT1/AMPK signaling pathway after spinal cord injury. This study is the first to prove that resveratrol suppresses neuronal apoptosis, reduces tissue injury, and promotes recovery of motor function via the activation of SIRT1/AMPK autophagy signaling pathway. These results offer a novel molecular mechanism for the neuroprotective effects

of resveratrol and potential clinical application of resveratrol to SCI therapy. However, this study only focused on the role of resveratrol in autophagy after SCI. As a natural polyphenolic compound, resveratrol exhibits a multitude of beneficial health properties including anti-oxidant, anti-inflammatory, and anti-tumor effects (Kraft et al. 2009; Kalantari and Das, 2010; Fernández and Fraga, 2011; Feng et al. 2016). The other potential mechanisms of resveratrol on SCI will be further studied. Acknowledgement This work was supported by the National Natural Science Foundation of China (NSFC) (Nos. 81471854; Nos. 81671907; Nos. 81601727). I would like to express my heartfelt gratitude to all co-authors for their assistance in completing this study and writing this paper. Abbreviations AMPK, Adenosine 5′ monophosphate-activated protein kinase; Bax, Bcl-2 associated X protein; BBB, Basso, Beattie, and Bresnahan; DMEM, Dulbecco's Modified Eagle Medium; DMSO, dimethyl sulfoxide; HE, hematoxylin and eosin ; LPS, lipopolysaccharides; PBS, phosphate buffer saline ; PFA, paraformaldehyde ; RT, room temperature; RT-PCR, reverse transcriotion-polymerase chain reaction; SD, Sprague–Dawley; SIRT1, Sirtuin 1; TBST, Tris-buffered Saline with Tween 20 References

Allen AR. (1911). Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column: a preliminary report. Journal of the American Medical Association. 57:878-80. Amar, A. P., & Levy, M. L. (1999). Pathogenesis and pharmacological strategies for mitigating secondary damage in acute spinal cord injury.Neurosurgery, 44(5), 1027-1039. Apel, A., Herr, I., Schwarz, H., Rodemann, H. P., & Mayer, A. (2008). Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy. Cancer Research, 68(5), 1485-1494. Apfeld, J., O'Connor, G., Mcdonagh, T., Distefano, P. S., & Curtis, R. (2004). The amp-activated protein kinase aak-2 links energy levels and insulin-like signals to lifespan in c. elegans. Genes & Development,18(24), 3004-9. Bao, Y. Y., University, F. M. M., & Guangzhou. (2003). Effects of resveratrol on secondary damages after acute spinal cord injury in rats.Acta Pharmacologica Sinica, 24(7), 703-10. Basso, D. M., Beattie, M. S., & Bresnahan, J. C. (1995). A sensitive and reliable locomotor rating scale for open field testing in rats. Journal of Neurotrauma, 12(1), 1-21.

Bastianetto, S., Menard, C., & Quirion, R. (2014). Neuroprotective action of resveratrol. biochim biophys acta. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1852(6), 1195-1201. Bellaver, B., Souza, D. G., Souza, D. O., & Quincozes-Santos, A. (2014). Resveratrol increases antioxidant defenses and decreases proinflammatory cytokines in hippocampal astrocyte cultures from newborn, adult and aged wistar rats. International Journal of Climatology,28(4), 479-84. Berggren, L. P. O. (2006). Sirt1: a metabolic master switch that modulates lifespan. Nature Medicine, 12(1), 34-36. Cantó, C., Gerhart-Hines, Z., Feige, J. N., Lagouge, M., Noriega, L., & Milne, J. C., et al. (2009). Ampk regulates energy expenditure by modulating nad+ metabolism and sirt1 activity. Nature, 458(7241), 1056-1060. Criollo, A., Senovilla, L., Authier, H., Maiuri, M. C., Morselli, E., & Vitale, I., et al. (2010). Ikk connects autophagy to major stress pathways.Autophagy, 6(1), 189-91. Degenhardt K; Mathew R; Beaudoin B; Bray K; Anderson D; Chen G; Mukherjee C; Shi Y; Gélinas C; Fan Y; Nelson DA; Jin S; White E. (2006). Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell, 10(1), 51-64.

Denu, J. M. (2012). Fortifying the link between sirt1, resveratrol, and mitochondrial function. Cell Metabolism, 15(5), 566-7. Donnelly LE, Newton R, Kennedy GE, Fenwick PS, Leung RH, Ito K, Russell RE, Barnes PJ. (2004). Anti-inflammatory effects of resveratrol in lung epithelial cells: molecular mechanisms. Am J Physiol Lung Cell Mol Physiol 287:L774–83 Feng Y, Cui Y, Gao JL, Li R, Jiang XH, & Tian YX, et al. (2016). Neuroprotective effects of resveratrol against traumatic brain injury in rats: involvement of synaptic proteins and neuronal autophagy. Molecular medicine reports. Fernández, A. F., & Fraga, M. F. (2011). The effects of the dietary polyphenol resveratrol on human healthy aging and lifespan. Epigenetics,6(7), 870-4. Galluzzi, L., Morselli, E., Vicencio, J. M., Kepp, O., Joza, N., & Tajeddine, N., et al. (2008). Life, death and burial: multifaceted impact of autophagy. Biochemical Society Transactions, 36(5), 786-90. Galluzzi, L., Vicencio, J. M., Kepp, O., Tasdemir, E., Maiuri, M. C., & Kroemer, G. (2008). To die or not to die: that is the autophagic question.Current Molecular Medicine, 8(2), 78-91. Gurusamy, N., Lekli, I. S., Ray, D., Ahsan, M. K., Gherghiceanu, M., & Popescu, L. M., et al. (2010). Cardioprotection by resveratrol: a novel

mechanism via autophagy involving the mtorc2 pathway. Cardiovascular Research, 86(1), 103-112. Gut, P., & Verdin, E. (2013). Rejuvenating sirt1 activators. Cell Metabolism, 17(5), 635-7. He, M., Ding, Y., Chu, C., Tang, J., Xiao, Q., & Luo, Z. G. (2016). Autophagy induction stabilizes microtubules and promotes axon regeneration after spinal cord injury. Proceedings of the National Academy of Sciences of the United States of America, 113(40). Hou, X., Xu, S., Maitland-Toolan, K. A., Sato, K., Jiang, B., & Ido, Y., et al. (2008). Sirt1 regulates hepatocyte lipid metabolism through activating amp-activated protein kinase. Journal of Biological Chemistry, 283(29), 20015-26. Ido, Y., Duranton, A., Lan, F., Weikel, K. A., Breton, L., & Ruderman, N. B. (2014). Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the ampk-foxo3 cascade in cultured primary human keratinocytes. Plos One, 10(2). Isaac, L., & Pejic, L. (1995). Secondary mechanisms of spinal cord injury. Surgical Neurology, 43(5), 484-5. Kalantari, H., & Das, D. K. (2010). Physiological effects of resveratrol.Biofactors, 36(5), 401-6.

Kesherwani, V., Atif, F., Yousuf, S., & Agrawal, S. K. (2013). Resveratrol protects spinal cord dorsal column from hypoxic injury by activating nrf-2.Neuroscience, 241(26), 80-88. Kraft, T. E., Parisotto, D., Schempp, C., & Efferth, T. (2009). Fighting cancer with red wine? molecular mechanisms of resveratrol. Critical Reviews in Food Science & Nutrition, 49(9), 782-99. Kroemer, G. (2010). Cross talk between apoptosis and autophagy by caspase-mediated cleavage of beclin 1. Oncogene, 29(12), 1717–1719. Lastra, C. A. D. L., & Villegas, I. (2005). Resveratrol as an anti-inflammatory and anti-aging agent: mechanisms and clinical implications.Molecular Nutrition & Food Research, 49(5), 405–430. Li, H. T., Zhao, X. Z., Zhang, X. R., Li, G., Jia, Z. Q., & Sun, P., et al. (2016). Exendin-4 enhances motor function recovery via promotion of autophagy and inhibition of neuronal apoptosis after spinal cord injury in rats. Molecular Neurobiology, 53(6), 1-10. Lin, H., Tang, H., Davis, F. B., & Davis, P. J. (2011). Resveratrol and apoptosis. Annals of the New York Academy of Sciences, 1215(1), 79-88. Lipinski, M. M., Wu, J., Faden, A. I., & Sarkar, C. (2015). Function and mechanisms of autophagy in brain and spinal cord trauma. Antioxidants & Redox Signaling, 23(6), 565-77.

Liu, C., Shi, Z., Fan, L., Chen, Z., Wang, K., & Bo, W. (2011). Resveratrol improves neuron protection and functional recovery in rat model of spinal cord injury. Brain Research, 1374(3), 100-9. Lou, J., Lenke, L. G., Ludwig, F. J., & O'Brien, M. F. (1998). Apoptosis as a mechanism of neuronal cell death following acute experimental spinal cord injury. Spinal Cord, 36(10), 683-90. Lu, J., Ashwell, K. W., & Waite, P. (2000). Advances in secondary spinal cord injury: role of apoptosis. Spine, 25(14), 1859-66. Maiuri, M. C., Criollo, A., & Kroemer, G. (2010). Crosstalk between apoptosis and autophagy within the beclin 1 interactome. Embo Journal,29(3), 515-6. Medina, D. L., Di, P. S., Peluso, I., Armani, A., De, S. D., & Venditti, R., et al. (2015). Lysosomal calcium signalling regulates autophagy through calcineurin and tfeb. Nature Cell Biology, 17(3), 288-299. Milner, J., & Allison, S. J. (2011). Sirt1, p53 and mitotic chromosomes.Cell Cycle, 10(10), 3049. Mizushima, N., Levine, B., Cuervo, A. M., & Klionsky, D. J. (2008). Autophagy fights disease through cellular self-digestion. Nature,451(7182), : 1069–1075. Oyinbo, C. A. (2011). Secondary injury mechanisms in traumatic spinal cord injury: a nugget of this multiply cascade. Acta Neurobiologiae Experimentalis, 71(2), 281-99.

Salminen A, Kaarniranta K, & Kauppinen A. (2016). Age-related changes in ampk activation: role for ampk phosphatases and inhibitory phosphorylation by upstream signaling pathways. Ageing research reviews, 28, 15-26. Salminen, A., & Kai, K. (2012). Amp-activated protein kinase (ampk) controls the aging process via an integrated signaling network. Ageing Research Reviews, 11(2), 230-241. Satoh, A., & Imai, S. (2014). Hypothalamic sirt1 in aging. Aging, 6(1), 1-2. Sedding, D., & Haendeler, J. (2007). Do we age on sirt1 expression?.Circulation Research, 100(10), 1396-8. Shin, S. M., Cho, I. J., & Kim, S. G. (2009). Resveratrol protects mitochondria against oxidative stress through amp-activated protein kinase-mediated glycogen synthase kinase-3beta inhibition downstream of poly(adp-ribose)polymerase-lkb1 pathway. Molecular Pharmacology,76(4), 884-95. Shintani, T., & Klionsky, D. J. (2004). Autophagy in health and disease: a double-edged sword. Science, 306(5698), 990-5. Stenesen, D., Suh, J. M., Jin, S., Yu, K., Lee, K. S., & Kim, J. S., et al. (2013). Adenosine nucleotide biosynthesis and ampk regulate adult life span and mediate the longevity benefit of caloric restriction in flies. Cell Metabolism, 17(1), 101–112.

Tator, C. H., & Fehlings, M. G. (1991). Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. Journal of Neurosurgery, 75(1), 15-26. Toerge, J. E., Rosen, J. S., & Calenoff, L. (1978). Secondary spinal lesions in spinal cord injury patients. Archives of Physical Medicine & Rehabilitation, 59(7), 343-5. Udenigwe, C. C., Ramprasath, V. R., Aluko, R. E., & Jones Peter, J. H. (2008). Potential of reveratrol in anticancer and anti-inflammatory therapy.Nutrition Reviews, 66(8), 445-54. Wang, Y., Liang, Y., & Vanhoutte, P. M. (2011). Sirt1 and ampk in regulating mammalian senescence: a critical review and a working model. Febs Letters, 585(7), 986-94. Xiao, B., Sanders, M. J., Underwood, E., Heath, R., Mayer, F. V., & Carmena, D., et al. (2011). Structure of mammalian ampk and its regulation by adp. Nature, 472(7342), 230-3. Yun, H., Park, S., Kim, M. J., Yang, W. K., Dong, U. I., & Yang, K. R., et al. (2014). Amp-activated protein kinase mediates the antioxidant effects of resveratrol through regulation of the transcription factor foxo1.Febs Journal, 281(19), 4421-38. Zhang Yanlin, Zhu Wawa, Liu Zhihua, Liu Huihui, Zhou Yande, & Cao Yongjun, et al. (2016). Resveratrol enhances autophagic flux and

promotes ox-ldl degradation in huvecs via upregulation of sirt1:. Oxidative medicine and, cellular longevity, 2016(22), 1-13. Zhou, Y., Zhang, H., Zheng, B., Ye, L., Zhu, S., & Johnson, N. R., et al. (2016). Retinoic acid induced-autophagic flux inhibits er-stress dependent apoptosis and prevents disruption of blood-spinal cord barrier after spinal cord injury. International Journal of Biological Sciences, 12(1), 87-99. Figure Legends Figure 1 Resveratrol improves functional recovery following SCI in rats Locomotor recovery was assessed by BBB scoring before and after spinal cord contusion in rats treated with resveratrol or DMSO. Resveratrol treated rats had significantly higher BBB scores ON 14 and 28 days after SCI. Data presented as mean ± SD (n = 5 in each group. *p < 0.05, **p < 0.01 compared to vehicle group). Figure 2 Resveratrol reduces motor neuron loss and lesion size following SCI in rats Sections of spinal cord from spinal cord contused rats treated with resveratrol or DMSO underwent Nissl (A) or HE (B) staining 7 days after injury. There were significantly more motorneurons in the anterior horn of tissue from resveratrol treated rats than vehicle treated rats (C). Lesion size was significantly smalled in resveratrol-treated rat tissue than tissue from vehicle treated rats (D). Scale bars represent 100 and 500 μm, respectively. Data presented as mean ± SD (n=3 in each group. **p <

0.01 in comparison between groups). Figure 3 Resveratrol promotes protein expression of autophagy following SCI in rats Protein expression of SIRT1, p-AMPK, Beclin-1, LC3-B and p62 in spinal tissue from SCI rats treated with reservatrol or DMSO and sham rats 7 days after surgery was detected by Western blot (A). Expression of SIRT1 (B), p-AMPK (C), Beclin-1 (D) and LC3-B (E) was significantly higher in resveratrol treated rats than in rats receiving the vehicle. Expresion of p62 (F) was significantly inhibited in resveratrol treated animals. β-Actin served as an internal reference. Data presented as mean ± SD (n=4 in each group. *p < 0.05, **p < 0.01 in comparison between groups). Figure 4 Resveratrol promotes mRNA expression of autophagy following SCI in rats mRNA expression of SIRT1 and Beclin-1 in spinal tissue from SCI rats treated with resveratrol or DMSO and sham rats 7 days after surgery was detected by reverse transcription-polymerase chain reaction (RT-PCR) (A). mRNA expression of SIRT1 (B) and Beclin-1 (C) in rats treated with resveratrol was significantly higher in comparison to rats in sham and vehicle groups. Data presented as mean ± SD (n=4 in each group. *p < 0.05, **p < 0.01 in comparison between groups). Figure 5 Resveratrol elevates LC3-B expression following SCI in rats

The expression of LC3-B in spinal tissue from SCI rats treated with resveratrol or DMSO and sham rats 7 days after surgery was detected by immunofluorescence analysis (A). Expression of LC3-B protein in the presence of resveratrol was significantly higher when compared with vehicle treatment rats (B). Scale bars represent 50 and 100 μm, respectively. Data presented as mean ± SD (n=3 in each group. **p < 0.01 in comparison between groups). Figure 6 Resveratrol inhibits protein expression of apoptosis following SCI in rats The expression levels of Cleaved Caspase-3, Caspase-9, Bcl-2, and Bax protein were detected using Western blot analysis 7 days after spinal cord injury (A) . Cleaved Caspase-3, Caspase-9, and Bax levels were higher and Bcl-2 levels were lower in rats receiving vehicle (DMSO) when compared with sham rats after SCI. However, in comparison with vehicle rats, resveratrol treatment significantly reduced expression of Cleaved Caspase-3 (B), Caspase-9 (C), and Bax (E), and conversely increased the expression of Bcl-2 (D). β-Actin served as an internal reference. Data presented as mean ± SD (n=4 in each group. *p < 0.05, **p < 0.01 in comparison between groups). Figure 7 Resveratrol inhibits Bax expression and enhances Bcl-2 expression following SCI in rats The expression levels of Bcl-2 and Bax mRNA were detected using

reverse transcription-polymerase chain reaction (RT-PCR). Bax mRNA expression was significantly lower (A) in resveratrol-treated rats in comparison with vehicle rats while Bcl-2 mRNA expression was markedly higher (B). Data presented as mean ± SD (n=4 in each group. **p < 0.01 in comparison between groups). Figure 8 Resveratrol inhibits Cleaved Caspase-3 expression following SCI in rats The expression of Cleaved Caspase-3 in spinal tissue from SCI rats treated with resveratrol or DMSO and sham rats 7 days after surgery was detected by immunofluorescence analysis (A). The expression of Cleaved Caspase-3 protein in the presence of resveratrol was significantly lower when compared with the vehicle treated group (B). Scale bars represent 100 μm. Data are presented as mean ± SD (n=3 in each group. **p < 0.01 in comparison between groups). Figure 9 Resveratrol upregulates autophagy and inhibits apoptosis after SCI in rats The association of autophagy upregulation and block of apoptosis by resveratrol treatment after SCI was detected by coimmunostaining of p62 and Cleaved Caspase-3 (A). Expression of p62 protein in sham group was significantly lower when compared with vehicle treatment rats, while expression of Cleaved Caspase-3 showed remarkable upregulation. In comparison between vehicle group and resveratrol group, both p62 and

Cleaved Caspase-3 decreased significantly (B). Scale bars represent 100 μm. Data are presented as mean ± SD (n=3 in each group. **p < 0.01 in comparison between groups). Figure 10 LC3-B expression was inhibited by SIRT1 and AMPK inhibitors in primary neurons. The

expression

of

LC3-B

in

primary

neurons

treated

with

lipopolysaccharides (LPS) alone, LPS and reseveratrol, or LPS and reseveratrol in combination with either Compound C, EX527 or neither was detected by immunofluorescence analysis (A). The expression of LC3-B protein in the presence of resveratrol increased significantly compared with the vehicle treatment. However, in Compound C and EX527 groups, the expression level of LC3-B showed significant decrease in comparison with resveratrol treatment. Scale bars represent 50 and 100 μm, respectively. Data are presented as mean ± SD (n=3 in each group. **p < 0.01 in comparison between groups). Figure 11 TUNEL expression was inhibited by SIRT1 and AMPK inhibitors in primary neurons. The

expression

of

TUNEL in

primary

neurons

treated

with

lipopolysaccharides (LPS) alone, resveratrol alone, or LPS and reseveratrol in combination with either Compound C (SIRT1 inhibitor), EX527 (AMPK inhibitor) or neither was detected by TUNEL staining. The proportion of TUNEL positive neurons in resveratrol treated cells

was significantly lower when compared to untreated cells. However, the percentage of TUNEL positive neurons was significantly higher in cells treated with Compound C or EX527 in comparison with resveratrol treated cells (B). Scale bars represent 100 μm. Data are presented as mean ± SD (n=3 in each group. **p < 0.01 in comparison between groups).

Highlights Resveratrol treatment ameliorates rehabilitation of motor function after

spinal cord injury. Resveratrol increases the survival of motor neurons and reduces the size of injury after spinal cord injury. Resveratrol up-regulates SIRT1/AMPK signaling pathway and promotes autophagy after spinal cord injury. Resveratrol inhibits apoptosis in spinal cord injury rats. Inhibition of SIRT1/AMPK signaling pathway decreases expression level of autophagy and increases apoptosis.