Hydrogen sulfide protects spinal cord and induces autophagy via miR-30c in a rat model of spinal cord ischemia-reperfusion injury
© Li et al. 2015
Received: 29 December 2014
Accepted: 16 April 2015
Published: 7 July 2015
Hydrogen sulfide (H2S), a novel gaseous mediator, has been recognized as an important neuromodulator and neuroprotective agent in the nervous system. The present study was undertaken to study the effects of exogenous H2S on ischemia/reperfusion (I/R) injury of spinal cord and the underlying mechanisms.
The effects of exogenous H2S on I/R injury were examined by using assessment of hind motor function, spinal cord infarct zone by Triphenyltetrazolium chloride (TTC) staining. Autophagy was evaluated by expressions of Microtubule associated protein 1 light chain 3 (LC3) and Beclin-1 which were determined by using Quantitative Real-Time PCR and Western blotting, respectively.
Compared to I/R injury groups, H2S pretreatment had reduced spinal cord infarct zone, improved hind motor function in rats. Quantitative Real-Time PCR or Western blotting results showed that H2S pretreatment also downregulated miR-30c expression and upregulated Beclin-1 and LC3II expression in spinal cord. In vitro, miR-30c was showed to exert negative effect on Beclin-1 expression by targeting its 3’UTR in SY-SH-5Y cells treated with Oxygen, Glucose Deprivation (OGD). In rat model of I/R injury, pretreatment of pre-miR-30c or 3-MA (an inhibitor for autophagy) can abrogated spinal cord protective effect of H2S.
H2S protects spinal cord and induces autophagy via miR-30c in a rat model of spinal cord hemia-reperfusion injury.
KeywordsAutophagy Beclin-1 miR-30c Microtubule associated protein 1 light chain 3 (LC3) Oxygen glucose deprivation (OGD)
Ischemia/reperfusion (I/R) injury of the spinal cord is a dynamic process that frequently occurs during a variety of clinical situations such as thoracoabdominal aortic surgery or spinal cord injury . Due to complicated pathogenic factor, such as, membrane permeability variation, fluid and electrolyte disorders, lipid oxidation, it is difficult to achieve effective treatment for spinal cord injury induced by I/R .
Hydrogen sulfide (H2S), endogenously derived from L-cysteine in several organs, such as brain, heart, kidney and liver , has been regarded as the third gasotransmitter and endogenous neuromodulator and plays multiple roles in the central nervous system under physiological and pathological states, especially in secondary neuronal injury. For example, H2S has been reported to protect brain from I/R injury through maintaining mitochondrial function, inhibiting proinflammatory factors, neutralizing ROS and reducing apoptosis . However, the protective effect of H2S on I/R injury of spinal cord has not been described. Thus, we attempt to investigated the possible regulation role of H2S on spinal cord I/R injury in vitro or in vivo. The treatment with H2S could be achieved by inhalation of H2S or administration of NaHS by intravenous injection. However, the study showed that it is difficult to control the concentration of inhaled H2S, which could result in toxicity to animals . Now, administration of NaHS by intravenous injection becomes the common treatment for I/R injury due to accuracy of concentration .
Micro-RNAs (miRNAs) are small noncoding regulatory RNAs of about 19–22 nucleotides that function as translational inhibitor or mRNA degradation promoter by binding to the 3’untranslated region of their target mRNAs . Recently, the biological roles of miRNA has been considered to be extensively involved in the organic I/R injury. For example, Bijkerk et al. miR-126 protect renal ischemia/reperfusion injury by promoting vascular integrity . miR30a/p53 axis plays an important role in triiodothyronine preventing cardiac I/R mitochondrial impairment and cell loss . Although no evidence for miR-30c in spinal cord I/R injury, it has been proved to be a neural protector by targeting brain derived neurotrophic factoras well as neurotrophin signaling and axon guidance . Based on miRNA being involved in H2S protected against ischemic injury , we speculates that miR-30c might be involved in H2S protecting I/R injury.
In addition, several miRNAs have also been reported to be involved in autophagy modulation by regulating the expression of autophagy-related genes . Autophagy, a regulated process of degradation and recycling of cellular constituents, can participated in organelle turnover and the bioenergetic management of starvation of spinal cord injury . During autophagy, part of the cytosol or entire organelles are sequestered into autophagosomes. Autophagosomes ultimately fuse with lysosomes, thereby generating single-membraned autophagolysosomes and degrading their content . In experiments, Beclin 1, the mammalian homologue of yeast Atg6, was first described as a Bcl-2-interacting protein , and its mediated autophagy plays an important role in the regulation of cell survival and death . Additionally, microtubule-associated protein 1 light chain 3 (LC3) were previously found to promote autophagy by transforming LC3-I into LC3-II. Notably, it has been reported the early and sustained activation of autophagy after spinal cord injury  and the change in expression of these autophagy-related proteins has also been recognized in rat model of spinal cord injury . Recently, autophagy has been reported to contributed to the protective effect of H2S against I/R injury in hepar  and colon epithelial cells . Thus, we attempted to testify whether that occurs in regulation effect of H2S on spinal cord I/R injury.
In this study, we established rat model of spinal cord ischemia/reperfusion injury and investigated protective effects of H2S and the underlying mechanism.
Chemicals and reagents
Ham’s F12 medium was obtained from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA). Lipofectamine 2000 transfection reagent was obtained from Invitrogen Life Technologies (Grand Island, NY, USA). NaHS was purchased from Sigma Aldrich (St. Louis, MO). Antibodies against Beclin-1, LC3-I, LC3-II and β-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Detergent Compatible (DC) Protein Assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
Animals and surgery
This study was performed in accordance with the National Institute of Health guidelines for the use of experimental animals, and all animal protocols were approved by the Institutional Animal Care and Use Committee of China Medical University. Male Sprague-Dawley rats (China Medical University Animal Center, Shenyang China) weighting 300 to 350 g were used in the study. The rats were housed in a temperature-controlled environment under a light-dark cycle condition with free access to food and water.
Rat models of spinal cord ischemia-reperfusion injury were performed. Briefly, rats were intraperitoneally anesthetized with 10 % chloral hydrate (300 mg/kg). Rectal temperature was monitored and body temperature was maintained at 37.0 ± 0.5 °C with an infrared heat lamp and a heating pad. A 24-gauge catheter was inserted into the tail artery to monitor the mean distal arterial pressure (MDAP). Spinal cord ischemia was induced by inserting a 2 F-Fogarty balloon catheter (Edwards Life Science, Shanghai, China) through the left femoral artery into the proximal descending thoracic aorta. The distance between the site of insertion to the proximal descending thoracic aorta was approximately 11 cm. The balloon was inflated with 0.05 ml of distilled water to induce spinal cord ischemia. The efficiency of the occlusion was evidenced by an immediate and sustained loss of detectable pulse pressure and a decrease in MDAP.
Twelve hours after ischemia, the balloon was deflated, and spinal cord blood flow was restored. The arterial catheters were then removed, the incisions were sutured. The animals were cultured in appropriate environment for recover. Bladder content was compressed manually as required. Arterial blood samples were collected before ischemia 1 h and 6 h after the beginning of reperfusion, respectively.
The rats were randomly divided into three groups. The sham group (n = 10) underwent the surgical procedure but without aortic occlusion; The I/R injury group (n = 10) underwent the surgical procedure and was administered with an equal volume of saline; The I/R + NaSH group (n = 10) was intraperitoneally injected with NaSH (30 umol/kg, dissolved in saline) 30 minutes before the initiation of ischemia. Forty–eight hours after reperfusion, animals were anesthetized with 3 % halothane and euthanized by transcardiac perfusion with 100 mL of 0.9 % saline solution and 50 mL of 4 % paraformaldehyde in phosphatebuffered saline (PBS).
Assessment of neurologic function
The hind limb function was scored by the means of the Basso, Beattie, and Bresnahan (BBB) open-field locomotor scale, which ranges from 0 (no detectable hind limb movement) to 21 (normal hind limb locomotion) . Before surgery the rats were placed individually in a molded plastic open field to ensure that all subjects consistently obtained a maximum score of 21. BBB scores were recorded at 1, 6, 12, 24, and 48 hours in the acute phase after reperfusion by five experienced investigators who had not conducted the surgery and were unaware of the group treatments. The final score for each animal was determined by averaging the values from all investigators.
Staining with triphenyltetrazolium chloride
The ischemic infarct was exhibited by triphenyltetrazolium chloride (TTC) staining and evaluated by computer-aided image acquisition. Briefly, the I/R injured spinal cord were harvested, and sliced into 2.0-mm transverse sections. The slices were incubated in 2 % TTC (dissolved in PBS) for 30 minutes at 37 °C and then were stored in a 10 % formalin solution. Infarct areas were measured by using NIH Image (National Institutes of Health, Bethesda, Md) image analysis software, and data were represented as the percentage of the infarct area in the total area of the spinal cord section.
Quantitative Real-Time PCR
Total RNA was extracted from the spinal cord tissue or SY-SH-5Y cells with the Trizol Reagent (Invitrogen, Carlsbad, CA, USA), respectively. The mature miR-30(a–e), Beclin-1 mRNA and LC3 miRNA were quantified by using Quantitative Real-Time PCR (Q-RT-PCR) assays with fluorescent nucleic acid dye. Each sample (1 μg) was reverse-transcribed into cDNA by using the RealMasterMix First Strand cDNA Synthesis Kit (Tiangen) according to manufacture’s protocol. Real-time PCR was conducted by using SYBR Premix ExTagTM (Takara) according to the manufacturer’s protocols and performed in the Applied Biosystems 7500 Real-time PCR system. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. The miRNA expression levels were normalized to U6 RNA and the Beclin-1 and LC3 mRNA levels were normalized to actin mRNA. All reactions were run in triplicate.
Cells and oxygen, glucose deprivation (OGD) treatment
SY-SH-5Y cell line, derived from human hippocampal neurons, was obtained from Sigma-Aldrich (St. Louis, USA) and was cultured in Ham’s F12 medium including EMEM (EBSS) (1:1), 2 mM Glutamine, 1 % Non Essential Amino AcidsNEAA, 15 % Foetal Bovine Serum FBS/FCS at 37 °C. The neuronal injury during ischemia is due to a reduction in the supplement of oxygen and glucose that is named as Oxygen, Glucose Deprivation (OGD). The OGD experiment in vitro is thought to mimic the pathological conditions of ischemia. In the present study, OGD was conducted as described previously  in neurons. OGD solution (pH 7.4) containing (in mM): 20 NaHCO3, 120 NaCl, 5.36 KCl, 0.33 Na2HPO4, 0.44 KH2PO4, 1.27 CaCl2, 0.81 MgSO4. Was used. The OGD procedure is just as follows: Neurons were rinsed twice with OGD solution and were incubated in OGD solution in a hypoxic incubator (model 3131, Thermo Forma, San Jose, CA, USA) containing 94 % N2, 1 % O2 and 5 % CO2 for 1 hour; For reoxygenation (Reox), the neurons were incubated in OGD solution containing 5.5 mM glucose at 37 °C in a humidified 5 % CO2 atmosphere for 24 hours. NaSH (10, 100 and 200 μmol/l) was continuously administrated from 1 hour before OGD to the end of the experiment. The same procedure was carried out in the control neurons except the OGD treatment.
Bioinformatics analysis of miR-545 target in Ku70
Based on bioinformatics analysis, we predicted that hsa-miR-30c can bind with the 3’UTR region of Beclin-1 by using four common websites (Target Scan: http://www.targetscan.org/, miRanda: http://www.microrna.org/, Microcosm: http://www.ebi.ac.uk/enright- srv/microcosm/ cgi-bin/targets/v5/genome.pl, and PITA: http://genie.weizmann.ac.il/) (Fig. 4a).
Plasmid construction and luciferase reporter assays
For Beclin-1 3’UTR reporter assay, SY-SH-5Y cells were placed in 24-well plates (1 × 103 cells per well) and then transfected with either psi-CHECKTM2-WT-BECN-3’UTR (wild type) or psi- CHECKTM2-MT-BECN-3’UTR that containing the miR-30c targeting sequence (UGUUUAC) (mutant) dual Luciferase reporter plasmid (Promega, WI, USA) according to manufacture’s protocol. The mimics and inhibitors of hsa-miR-30c and their negative controls (RIBO Bio, Guangzhou, P.R. China) were cotransfected with the reporter plasmids at a final concentration of 100 nmol/μl. Forty–eight hours after transfection in neurons, luciferase activity in lysates was measured with a Dual-Luciferase Reporter Assay System (Promega, WI, USA) followed by the manufacture’s suggestions and normalized against the activity of the pRL-SV40. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega, Madison, WI), and the Renilla luciferase activity was normalized to firefly luciferase activity. Independent triplicate experiments were performed for each plasmid construct.
MTT proliferation assay
SY-SH-5Y cells were seeded at a density of 1000 cells/well. On the next day, after NaSH (100 μmol/l) treatment for 1 hour, cells were treated with OGD. For cell viability assay, 50 mL of 5 mg/mL 3–(4,5–Dimethylthiazol–2-yl) 22,5-diphenyltetrazolium bromide (MTT) was added to wells for 4 h, and then the media were replaced with DMSO for analysis of optical density using a mQuant Microplate Spectrophotometer (BioTek, UK) at a wavelength of 540 nm. All independent analysis were run in triplicate.
Cell treatment with miR-30c inhibitor or mimics
SY-SH-5Y cells were treated with pre-miR-30c or anti-30c (Ambion Pre-miR miRNA Precursors, Life Technologies) using Oligofectamine (Life Technologies) according to the manufacturer’s instructions. miRNA mimics negative control (pre-NC) and anti-miRNA negative control (anti-NC) were severed as negative controls in the experiments respectively. Further analysis of the samples (infection or RNA isolation) was performed at 48 h post-transfection.
For analysis of protein expression, western blot assay was performed. Briefly, both tissues from the injured spinal cord (extending 2 mm to the infarct, minced with eye scissors) and cells were homogenized in lysis buffer (1 % NP–40, 50 mmol/L Tris, pH 7.5, 5 mmol/L EDTA, 1 % SDS, 1 % sodium deoxycholate, 1 % Triton X–100, 1 mmol/L PMSF, 10 mg/ml aprotinin, and 1 mg/ml leupeptin) and clarified by centrifuging for 20 min in a microcentrifuge at 4 °C. respectively. The total protein concentration was determined with the BCA Protein Assay Kit and the resulting supernatant (50 mg of protein) was subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The separated proteins were transferred to PVDF (Millipore) followed by blocking with 5 % nonfat milk and incubated with primary antibody against Beclin–1 (1:500 ), LC3–I (1:1000), LC3–II (1:1000) or β–actin (1:10000). The target proteins were captured by anti-rabbit horseradish peroxidase-conjugated secondary antibody. Finally, protein was visualized using an enhanced chemiluminescence system (ECL, Pierce Company, USA).
All statistical analyses were performed using SPSS 16.0 software (Chicago, IL). Student’s t test was used to analyze the significance between the two groups. One-way Analysis of Variance (ANOVA) was used to test the significance of the Beclin-1 3’UTR. BBB scores data were analyzed by two factor repeated measures analysis of variance (ANOVA; for group and time) followed by post hoc Bonferroni’s test for multiple comparisons. Error bars represent SD. P-Values < 0.05 were considered statistically significant.
H2S improved I/R-induced spinal cord injury
H2S promoted autophagy in rat model of spinal cord I/R injury
H2S improved OGD-induced ischemic injury in SY-SH-5Y cells
miR-30c acts as a negative regulator of Beclin-1 expression
To further demonstrate the effect of miR-30c on Beclin-1 expression, we transfected SY-SH-5Y cells with pre-miR-30c, anti-30c or their negative control, and then examined the expression of Beclin-1 in cells treated with OGD. Fig. 4c showed that treatment with pre-miR-34c suppressed Beclin-1 mRNA expression under ischemia, however, that was increased by cells treated with anti-30c under both ischemia and normoxia. Additionally, we attempted to detected the possible regulated LC3 by miR-30c and the result showed that the LC3 expression varied as Beclin-1 (Fig. 4d) indicating the key regulated role of miR-30c in OGD-induced cells autophagy.
S2H inhibited Beclin-1 3’UTR under OGD
Spinal core protective effect of H2S was reversed by miR-30c overexpression
Spinal cord ischemia/reperfusion injury, which occur in secondary neuronal injury after I/R spinal cord injury, is considered as a major health problem that is a frequent cause of disability and death and makes considerable demands on health services. Nevertheless, there was limited advance in the therapeutic strategies to counter spinal cord injury. Thus, neuroprotection and neurorecovery are still the main therapeutic strategies under development. An ideal neuroprotectant would be non-toxic, easily administered, and offer protection at all stages of injury, including prophylaxis. In this study, we exogenously treated spinal cord I/R injury rat with H2S, which acted as a novel signaling molecule in central nervous system , by injected with NaSH, and examined the function of spinal cord after injury. Our results showed that NaSH treatment improved the hide motor function and infarct zone in rat model of spinal cord I/R injury. The underlying mechanism is that administration of H2S activates autophagy that indicated by upregulation the expression of autophagy-related proteins including Beclin-1 and LC3II via inhibiting miR-30c expression after spinal cord reperfusion injury.
The time profile of BBB scoring system for hindlimb locomotor function after I/R injury was determined, and time effects of H2S were detected in this study. We observed that pretreatment of NaHS 30 min before I/R injury can alleviate I/R induced spinal cord injury in a manner of time dependent from hour 6 h to hour 48. In fact, previous studies demonstrated that pathological development of I/R results in neuronal cell death . Therefor, after 48 h I/R, measurement of infarct zone was performed in spinal cord tissues. We found in the present study that I/R obviously increased infarct zone that was effectively ameliorated by pretreatment 30 min before I/R injury by NaHS suggesting reducing I/R-induced neural cell lost may be contributed to the neuroprotective effects of H2S. These results was further demonstrated by result that H2S function as a neuroprotective factor for hippocampal neurons that treated by OGD. These findings indicate that H2S may be a potential therapy for spinal cord I/R injury.
Increased autophagy is observed in multiple and distinct experimental models of spinal cord injury [16, 25]. Autophagy plays an important roles in the stability of the body metabolism via affecting the mechanism of apoptosis as well as autophagic cell death. Previous sthudy showed that autophagy is important for the balance between protein synthesis and degradation in spinal cord injury . For example, in cases in which mitochondria are extensively damaged, autophagy may be protective by sequestering and degrading defective mitochondria before they can release death-inducing proteins . Notably, in the early nerve damage, including I/R injury autophagy process is activated to protect nerve cell against injury . This is also observed in our present study. The result of NaSH treatment obviously promoting expression of the LC3II and Beclin-1 which acted as markers for autophagy suggested that H2S protect spinal cord against I/R injury via increased neurocyte autophagy. It was supported by result of inhibition of autophagy by 3-MA abrogated H2S functional outcome after I/R injury in rats.
In addition, we confirmed and are the first to note that the miR-30c was downregulated during spinal cord protective effect of H2S in I/R injury. Previously, several microRNAs have also been reported to be involved in autophagy modulation by regulating the expression of autophagy-related genes [29, 30]. Recent study showed that miR-30 could impair autophagic process by targeting multiple genes in the autophagy pathway  . Thus, we hypothesized that there exists the possible targeting region of Beclin-1 mRNA by miR-30c, and it was supported by the result of bioinformatics analysis in Fig. 4a. To confirm this cellular metabolism, hippocampal neurons was transfected with pre-miR-30c or anti-30c to examined expression of Beclin-1. The data indicated the negative regulation of miRNA or protein expression of Beclin-1 by miR-30c.
The present study demonstrates that systemic administration of H2S improved spinal cord injury and motor function in rat model of I/R injury. H2S may serve as a neuroprotectant to treat I/R-induced spinal cord injury via activating autophagy in a miR-30c dependent signaling pathway. This founding might has potential clinical therapeutic value for treatment of spinal cord injury.
This work was supported by grants from the Natural Science Foundation of China (No. 81300130), Liaoning Science and Technology Project (No.2014021045)
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