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Moderate hypothermia protects increased neuronal autophagy via activation of extracellular signal-regulated kinase signaling pathway in a rat model of early brain injury in subarachnoid hemorrhage
Biochemical and Biophysical Research Communications 502 (2018) 338e344
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Moderate hypothermia protects increased neuronal autophagy via activation of extracellular signal-regulated kinase signaling pathway in a rat model of early brain injury in subarachnoid hemorrhage Junjie Liu a, b, Wenji Liang a, Jingyao Wang a, Yaning Zhao a, b, Yichao Wang a, Jingxi Zhang a, Jianmin Li a, b, * a b
College of Clinical Medicine, North China University of Science and Technology, Tangshan, 063000, PR China Department of Neurosurgery, Affiliated Hospital of North China University of Science and Technology, Tangshan, 063000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2018 Received in revised form 21 May 2018 Accepted 23 May 2018 Available online 30 May 2018
Moderate hypothermia (MH) used as treatment for neurological diseases has a protective effect; however, its mechanism remains unclear. Neuronal autophagy is a fundamental pathological process of early brain injury in subarachnoid hemorrhage (SAH). We found that moderate activation of autophagy can reduce nerve cells damage. In this study, We found that MH can moderately increase the level of autophagy in nerve cells and improve the neurological function in rats. This type of autophagy activation is dependent on extracellular signal-regulated kinase (ERK) signaling pathways. The level of neuronal autophagy was down-regulated significantly by using U0126, an ERK signaling pathway inhibitor. In summary, these results suggest that MH can moderately activate neuronal autophagy through ERK signaling pathway, reduce nerve cell death, and produce neuroprotective effects. © 2018 Elsevier Inc. All rights reserved.
Keywords: Subarachnoid hemorrhage Autophagy Moderate hypothermia Neuroprotection Extracellular signal-regulated kinase
1. Introduction SAH is a type of cerebrovascular disease that is caused by intracranial vascular rupture due to various factors and seriously threatens human health. The cure rate for SAH has increased significantly with the advances in surgical techniques and medical equipment. However, its morbidity and mortality are still high. The surviving patients also have different degrees of cognitive decline, obstacles and dysfunctions; their quality of life is negatively affected; and their communities and families are bestowed a heavy burden [1]. SAH may induce early brain injury (EBI) and delayed brain injury, which can manifest as cerebral edema, intracranial hypertension, cerebral infarction, neurological disorders, and disturbances of consciousness. These symptoms may result from inflammation, cerebral vasospasm, apoptosis, and autophagy [2]. However, the current pathogenesis and treatment strategy for SAH remain poorly understood and thus warrant further study. Hypothermia at 28e35 C was defined as moderate hypothermia (MH) [3]. MH has made great breakthroughs in basic and * Corresponding author. College of Clinical Medicine, North China University of Science and Technology, Tangshan, 063000, PR China. E-mail address: [email protected] (J. Li). https://doi.org/10.1016/j.bbrc.2018.05.158 0006-291X/© 2018 Elsevier Inc. All rights reserved.
clinical research. MH may exert neuroprotective effects by reducing the markers of apoptotic pathway in TBI rats [4]. MH can reduce the risk of vasospasm and DCI in patients with SAH, improve functional outcomes, and reduce mortality [5]. However, only few studies focused on the effects and molecular mechanisms of MH in SAH. Autophagy is a key homeostatic process wherein cytosolic proteins and organelles are degraded and recycled. This process maintains cellular homeostasis and survival [6]. As a clearance pathway, autophagy exerts protective effects in multiple neurological disease models [7]. Autophagy can be activated in the SAH model, and this activation has neuroprotective effects [8]. However, the mechanism of autophagy activation is complex and involves multiple signal pathways. The up-regulation of cell surface estrogen receptor alpha is associated with mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) activity and promotes autophagy maturation in vitro [9]. This study aims to investigate the effects of MH in EBI after SAH in rats. In particular, we elucidate whether or not treatment with MH after SAH would protect rats against EBI and would moderately activate neuronal autophagy. Possible underlying mechanism(s) of any actions are also analyzed.
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2. Materials and methods
2.4. Manipulation of temperature
2.1. Animals
Frontal cortex brain temperature was monitored with a digital electronic thermometer (model DP 80; Omega Engineering, Stamford, CT) and a 0.15 mm-diameter temperature probe (model HYP033-1-T-G-60-SMP-M; Omega Engineering), which was inserted 4.0 mm ventral to the surface of the skull. The probe was removed before FPI and replaced immediately after injury. Rectal temperatures were measured with an electronic thermometer with analog display (model 43 TE; YSI, Yellow Springs, OH) and a temperature probe (series 400; YSI). The 32 C brain temperature was achieved by immersing the anesthetized rat body in ice-cold water. The skin and fur of all 21 animals were protected from water by placing the animal in a plastic bag (head exposed) before immersion. Animals were removed from the water bath when the brain temperature was reduced to within 2 C of the target temperature. Approximately 30 min was required to reach the target brain temperatures, which were maintained for 4 h under general anesthesia in room temperature through the intermittent application of ice packs as needed. A brain temperature of 37 C was achieved under general anesthesia with a heating blanket.
The experimental protocol was approved by the Animal Care and Use Committee of Central North China science and Technology University of China (No. 2013-99) and has conformed to the standards of the Guide for Care and Use of Laboratory Animals established by the National Institutes of Health of the United State of America. All efforts were taken to minimize the number of rats used and their suffering. Adult males Sprague Dawley (SD) rats that weigh 350e450 g and are 10e12 weeks old were supplied by Vital River Laboratories Ltd., Beijing, China. The license number was SCXK (Beijing): 2015-003. The animals were fed and given free access to water during experiments conducted in the Center of North China Science and Technology University and were allowed to adapt to feeding 14 days before the experiment. Feeding environment: temperature 18e26 C, relative humidity 40e70%, and temperature in the general rat feeding box must be 1e2 C higher than that in the environment and must have high humidity ~10%. Noise was below 85 dB, and ammonia concentration was below 20 PPm. Light and shade with alternating light were provided every day. 2.2. Experimental design 2.2.1. Experiment I Rats were randomly divided into SHAM, SAH model, and MH þ SAH group to determine the neuroprotective effect of MH treatment. SAH model and MH þ SAH group were further divided into four subgroups: 6, 24, 48, and 72 h. The neurological function of rats was detected by Garcia score table, The brain edema index was detected by dry-wet method and HE staining. The morphology of neurons in hippocampus CA1 area was also observed. 2.2.2. Experiment II Rats were randomly split into three groups: SHAM, SAH model, and MH þ SAH group to observe the changes on autophagy-related protein Beclin-1 and LC3 after MH treatment. Beclin-1 and LC3 in the hippocampus of SAH rats were detected 24 h after SAH by using IHC and Western blot. 2.2.3. Experiment III Rats were randomly divided into four groups: SAH þ vehicle (equal amount of DMSO solution), MH þ SAH, U0126 þ SAH dissolved in DMSO at a dose of 0.05 mg/kg (R & D systems, Minneapolis, MN, USA), and U0126 þ MH þ SAH (dissolved in DMSO at a dose of 0.05 mg/kg) to evaluate the role of the ERK signaling pathway in activating autophagy induced by MH. The neurological function of rats was detected by Garcia score table and the brain edema index was detected in the hippocampus by dry-wet method. The expression of phosphorylated ERK1/2 (p-ERK1/2) Beclin-1 and LC3 were detected in the hippocampus by Western blot. 2.3. Establishing the SAH model The SAH model was induced by endovascular perforation. While the mouse was under pentobarbital (50 mg/kg) anesthesia, the left carotid artery and its branches were exposed and separated. The left external carotid artery was cut, and a 4-0 monofilament suture was advanced into the internal carotid artery through it until resistance was felt. Then, the suture was inserted further to puncture the vessel and to induce SAH. The sham rats underwent the same procedure but without vessel puncture.
2.5. Neurological score Neurological score were evaluated by two blinded investigator [10], the animals from each group were tested for their neurological function by a modified Garcia Scale method for spontaneous activity, limb symmetry, forearm extension, climbing response, body sensation, and responsive touch. The scores for each part were added to obtain the total score of neurological function. According to the scale, a low score indicates severe nervous system injury. On the contrary, a high score indicates less nerve damage. The lowest score is 3 and the highest score is 18. 2.6. Water content in brain The brain was separated into four parts, including the left hemisphere, right hemisphere, cerebellum, and brain stem. Each part was immediately weighed (wet weight) and promptly dried for 72 h at 105 C (dry weight). The water content was then measured [11]. 2.7. HE staining of rats' brains Rats were subjected to deep anesthesia by 10% chloral hydrate. All rats were perfused transcardially by 4% paraformaldehyde in phosphate-buffered saline (PBS). The brain was subsequently harvested, and the brain tissue spanning from the optic chiasm to the cerebral transverse fissure was resected. The tissue was embedded in paraffin, cut into coronal sections (5 mm), and stained with hematoxylin and eosin. Sections were observed under an optical microscope. The sections were cut in a microtome and allowed to adhere to glass slides with polylysine. Images of the ipsilateral hippocampus were captured at 400 by using a microscope (Nikon Labophot; Nikon USA, Melville, NY). Specimens were examined by two pathologists (blinded to group conditions) to identify cell death based on characteristic cellular morphological changes. Eight rats were included in each of the four groups. 2.8. IHC analysis of rats' brains The immunoreactivity of LC3 and Beclin-1 was detected by subjecting the 4 l m-thick formalin-fixed, OCT-embedded sections to IHC analysis. Endogenous peroxidase was blocked with 3% H2O2
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for 5 min, followed by a brief rinse in distilled water and a 15 min wash in PBS. The sections were cooled at room temperature for 20 min and then rinsed in PBS. Nonspecific protein binding was blocked by incubation in 5% horse serum for 40 min. The sections were incubated with primary antibodies (Abs; anti-LC3 [CST3868], anti-beclin-1 [CST3738], anti-p-ERK1/2 [ab1776660] and anti-IgG [immunoglobulin G], all diluted 1:300; boaosen Biological Co, Beijing, China) for 1 h at room temperature, followed by a 15 min wash in PBS. After being washed three times with PBS, the sections were stained with 3,30 -diaminobenzidine (DAB), counterstained with hematoxylin, dehydrated, cleared, and sealed. For negative controls, the sections were incubated in the absence of a primary Ab. Ten microscopic fields per each section were photographed randomly for the counting of LC3 and Beclin-1-positive cells (400 magnifications, Nikon TE300; Nikon). An investigator blinded to group conditions had collected all nonbiased stereological data for the study.
3.3. MH treatment reduces neuronal injury in hippocampal CA1 area SAH
2.9. Western blot analysis of rats' brains
3.4. MH treatment increases neuronal autophagy
Frozen brain samples were mechanically lysed in 20 mM Tris (pH 7.6) containing 0.2% sodium dodecyl sulfate (SDS), 1% Triton X100, 1% deoxycholate, 1 mM of phenylmethylsulphonyl fluoride, and 0.11 IU/Ml aprotinin (Sigma-Aldrich, St. Louis, Mo). Lysates were centrifuged at 12,000 g for 20 min at 4 C. Protein concentration was estimated by Bradford's method. Samples (60 lg/lane) were separated by 12% SDS polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% skimmed milk for 1 h at room temperature and incubated with primary Abs against LC3 and Beclin-1 (1:1000 dilutes both from CST, Inc., Danvers, MA) at 4 C overnight. Glyceraldehyde-3phosphate dehydrogenase (diluted 1:10,000; Sigma-Aldrich) was used as loading control. After being washed three times in Trisbuffered saline (TBS)þTween-20 (TBST) for 10 min each, the membrane was incubated in the appropriate horseradish-peroxidaseeconjugated secondary Ab (diluted 1:10,000 in skimmed milk) for 2 h and washed three times in TBST for 10 min each. The enhanced chemiluminescence kit (GE Healthcare, United States) was applied to generate chemiluminescent signals. All western blotting data were analysis was performed using ImageJ 6.0 software (National Institutes of Health, Bethesda, MD, United States).
Beclin-1 IHC staining was brown and was mainly expressed in the neuronal cell plasma. The Sham operation group showed occasional immune-positive cells and was lightly stained. Nerve cells were neatly arranged and had normal structure and nucleolus. The expression of SAH þ MH was significantly increased in the SAH group than in the Sham group. The former group was stained darker with many immunopositive cells. LC3 IHC staining was brown and was mainly expressed in the neuronal cell plasma. The Sham group showed occasional immune-positive cells and light staining. Its neurons were arranged in neat rows, had normal structure and clear nucleoli. Compared with that in the Sham group, immunity is significantly enhanced in the SAH group. Compared with the SAH group, SAH þ MH group was stained darker and had more immunopositive cells (Fig. 2A and B). Western blotting was performed in sham operation, SAH model, and HM þ SAH group after euthanizing the rats. Beclin-1 was detected at 24 h after SAH, and its expression in HM þ SAH group was significantly higher than that in SAH model group (1.25 ± 0.43 vs.1.48 ± 0.38, P < 0.05). LC3 was also detected at 24 h after SAH, and its expression in HM þ SAH group was significantly higher than that in SAH model group (1.34 ± 0.29 vs.1.61 ± 0.41, P < 0.05) (Fig. 2C, D, and 2E).
3. Results
3.5. Effect of inhibitor of ERK signaling on neurological function and cerebral edema index at 24 h in SAH group
3.1. MH treatment improves neurological function after SAH Fig. 1A and B shows that the sham operation group and the model group were successfully established. Compared with the sham operation group (17.8 ± 1.0), SAH group showed neurological impairment at each time point. Compared with those in the SAH model group, the neurological deficits in the MH þ SAH treated group were significantly relieved at all the time points and reached the highest level at 72 h (10.01 vs.14.85,P < 0.05) (Fig. 1C). 3.2. MH treatment reduces brain edema after SAH Compared with the sham operation group (78.3 ± 0.3), SAH group showed increased brain edema at each time point. Compared with those in the SAH model group, brain edema deficits in the MH þ SAH treated group were significantly relieved at all the time points, and the highest level (82.7 ± 0.4 vs.81.4 ± 0.1, P < 0.05) was observed at 24 h (Fig. 1D).
The neurons in the hippocampus CA1 area of Sham group were arranged neatly. The nucleus was positive, the nucleolus was clearly visible, the inclusion pulp was indifferent, and the axons were evenly seen. In the SAH group, the neurons were arranged relatively loosely. The hierarchical structure was disrupted, and the structure of the tissue was severely damaged. Many nucleus pyknosis and nucleolus disappeared. The surrounding tissue dissolved and thus showed the vascular components. Compared with the SAH group, MH þ SAH group showed significantly reduced neuronal damage, relatively mitigated cell edema, relatively neat structure, and less neuron death (Fig. 1E). Statistical analysis shows that the neuronal mortality in MH þ SAH group was significantly lower than that in SAH group and reach the lowest level (40.79% ± 1.9% vs.29.15% ± 3.6%, P < 0.05) at 6 h (Fig. 1F).
Compared with the Vehicle þ SAH group, MH þ SAH group showed neurological impairment (4.12 ± 1.3 vs. 6.57 ± 1.2, P < 0.05). Compared with that in the MH þ SAH group, the neurological impairment in U0126 þ SAH group was significantly reduced (6.57 ± 1.2 vs. 4.15 ± 0.9, P < 0.05). No significant difference in neurological score was detected between U0126 þ MH þ SAH and U0126 þ SAH groups (Fig. 3A). Compared with the Vehicle þ SAH group, MH þ SAH group showed reduced brain edema (82.50 ± 0.5 vs. 79.44 ± 1.2, P < 0.05). Compared with that in the MH þ SAH group, brain edema in the U0126 þ SAH group was increased significantly (79.44 ± 1.2 vs. 82.49 ± 0.8, P < 0.05). No significant difference in brain edema was detected between U0126 þ MH þ SAH and U0126 þ SAH groups (Fig. 3B). 3.6. Effects of ERK signaling pathway inhibitors on neuronal autophagy after 24 h in SAH group U0126 treatment significantly inhibited the expression of p-
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Fig. 1. Neuroprotective effect of moderate hypothermia (MH) on early brain injury (EBI) in rats with subarachnoid hemorrhage (SAH). (A) Bulk specimen sham of operation and model group; (B) IHC analysis shows of the sham operation and the model group under the microscope (200 ); (C) Garcia score of SHAM, SAH, and MH þ SAH group, at 6, 24, 48, and 72 h; (D) Brain water content in MH þ SAH and SAH group, at 6, 24, 48, and 72 h; (E) Damage on nerve cells in SHAM, SAH, and MH þ SAH groups (HE, 400 ); (F) Neuronal cell death rate in SHAM, SAH, and MH þ SAH groups was significantly lower than that in SAH group at 6, 24, 48, and 72 h. According to t-test, *P˂0.05, **P˂0.01. Values are mean ± SD (n ¼ 6 independent experiments).
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Fig. 2. Effect of MH on the expression of autophagy-related proteins Beclin-1 and LC3 in SAH rats. (A) IHC images of the localization and expression of Beclin-1 and LC3 in hippocampus CA1; (B) Results of quantitative analysis of IHC; (C) Western blot analysis of the Beclin-1 and LC3 expression in hippocampus at 24 h after SAH; (D) Quantification analysis of Beclin-1 expression in hippocampus at 24 h in SAH group; (E) Quantification analysis of LC3 expression in hippocampus at 24 h after SAH. According to t-test, ***p < 0.01, ***p < 0.001 vs. SHAM; ##p < 0.01 vs. SAH, Values are mean ± SD, (n ¼ 6 independent experiments).
ERK1/2, which was significantly down-regulated in U0126 þ SAH group compared with that in vehicle þ SAH group (0.65 ± 0.10 vs.0.25 ± 0.09, P < 0.0001). Compared with that in the Vehicle þ SAH group, the expression of ERK1/2 was significantly up-regulated in MH þ SAH group (0.65 ± 0.10 vs. 0.81 ± 0.08, P < 0.001). ERK1/2 expression was not significantly different between U0126 þ MH þ SAH and MH þ SAH groups (Fig. 3C and D). U0126 treatment significantly inhibited the expression of Beclin-1, which was significantly down-regulated in U0126 þ SAH group compared with that in vehicle þ SAH group (0.54 ± 0.11 vs.0.19 ± 0.09, P < 0.0001). Beclin-1 expression was not significantly different between U0126 þ MH þ SAH and MH þ SAH groups (Fig. 3C and E). U0126 treatment significantly inhibited the transformation from LC3-I to LC3-II. The expression of LC3-Ⅱ was significantly
down-regulated in U0126 þ SAH group compared that in vehicle þ SAH group (0.43 ± 0.09 vs. 0.18 ± 0.07, P < 0.001). Compared with that in Vehicle þ SAH group, the expression of LC3II in MH þ SAH group was significantly increased (0.43 ± 0.09 vs. 0.67 ± 0.12, P < 0.001). LC3-II expression was not significantly different in U0126 þ SAH and U0126 þ MH þ SAH groups (Fig. 3C and F). 4. Discussion In this study, we found that SAH causes hippocampal cell death, decreases neurological function, and increases mouse cerebral edema, which further peaked at 24 h post-SAH. MH could attenuate hippocampal cell death, reduce brain edema, and improve neurological function. We also found that autophagy increased after SAH
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Fig. 3. HM-affected autophagy is dependent on the ERK signaling pathway after SAH. (A) Neuroprotective effect of MH after U0126 treatment; (B) U0126 can aggravate the degree of brain edema after moderate hypothermia; (C) Western-blot analysis of the p-ERK1/ 2,Beclin-1 and LC3 expression in hippocampus at 24 h after SAH; (D) Quantification analysis of p-ERK1/2 expression in hippocampus at 24 h after SAH; (E) Quantification analysis of Beclin-1 expression in hippocampus at 24 h after SAH; (F) Quantification analysis of LC3 expression in hippocampus at 24 h after SAH. According to t-test, *p < 0.05, ***p < 0.001 vs. Vehicle, ###p < 0.05, ###p < 0.001 vs. MH, Values are mean ± SD (n ¼ 6 independent experiments).
and further increased after MH treatment. The results suggest that the autophagy moderate activation at EBI of SAH by MH may be a neuroprotective mechanism. However, the relationship of MH with autophagy is unknown. We detected the sensitive indicator of ERK signaling pathway P-ERK1/2 and found that the autophagy activation by MH is dependent on the ERK signaling pathway. MH refers to light and moderate low temperatures of 30e34 C. It was found that MH can significantly improve the prognosis of patients with brain injury [12]. MH is a feasible treatment and can
be safely used in patients with poor-grade SAH. In addition, this process may reduce the risk of vasospasm and DCI, thus improving the functional outcomes and reducing mortality [5]. However, the neuroprotective effect of MH on SAH is still poorly understood. Our study show that the neuroprotective effects of MH have been validated in the rat model of disease, MH treatment can significantly reduce neuronal death in the hippocampus of SAH rats and improve the neurological function scores. Therefore, we believe that MH has a neuroprotective effect on SAH.
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Induced Moderate autophagy activation is essential to maintain cellular homeostasis and cell survival. Experimental data show that autophagy has neuroprotective effect. Hispidulin significantly induces autophagy in H4 cells. This phenomenon contributes to its protective activity against sevoflurane-induced apoptosis [13]. These findings highlight that hispidulin offers neuroprotection against sevoflurane-induced cognitive dysfunction, which is mediated by autophagy. Liraglutide can promote autophagy, thereby reducing neuronal damage in the hippocampus and promoting cognitive recovery in diabetic cognitive disorder mice. Autophagy is known to have neuroprotective effects [14]. MH can increase autophagy in the hippocampal neurons and thus reduce brain damage in the TBI rat model [15]. However, whether or not the neuroprotective effect of MH on SAH can also increase the autophagy of the nerve cells remain unclear. Beclin-1 and LC3 are biomarkers for detecting changes in autophagy. We found that Beclin-1 and LC3 expression were up-regulated after MH treatment in rats. Therefore, we believe that the neuroprotective effect of MH on SAH rats is achieved by up-regulating the autophagy of nerve cells. ERK plays an important role in regulating autophagy. The ERK signal cascade is activated in several well established autophagy models, and inhibition of this pathway inhibits autophagy [16]. ERK signaling pathway inhibitor U0126 induces the expression of neurotoxin 1-methyl-4-phenylpyridinium (MPP) in damaged neurons, reduces the level of autophagy protein LC3 cells, and increases mitochondrial damage [17]. ERK1/2 activators stimulate the phosphorylation of Galpha-interacting protein (GAIP) to stimulate macrophages in human colon cancer cell line HT-29 [18]. Our experimental results showed that the expression of Beclin-1 and LC3 in the hippocampus of rats was significantly down-regulated. The damage of nerve cells was intensified after the U0126, ERK signaling pathway inhibitor was applied. The activation of autophagy was regulated by MEK/ERK signaling pathway in EBI of SAH. As a sensitive indicator of ERK signaling pathway, p-ERK1/2 was detected in this experiment. We found that the effect of MH on autophagy depends on the ERK signaling pathway. In this study, we observed that SAH could increase autophagy in the hippocampus, and MH could accelerate this process. Autophagy is the main mechanism for the bulk elimination of aberrant cell components and could interact with other cell death mechanisms. We found that MH could attenuate cell death in the ipsilateral hippocampus. Furthermore, this process relies on the ERK signaling pathway. Therefore, we propose that the neuroprotective effects of MH may be related to the interaction between autophagy and ERK signaling pathway. In conclusion, autophagy pathway as mediated by ERK signaling pathway may participate in the neuroprotective effect of post-SAH MH. However, further work will be required to reveal the possible upstream target molecules of the promotion of autophagy via ERK signaling pathway by MH. Author contribution Jianmin Li designed research; junjie Liu, yaning Zhao, Jingxi Zhang and Jingyao Wang performed research; Yichao Wang analyzed data; Wenji Liang wrote the paper. All authors reviewed
the manuscript. Acknowledgements This work was supported by the Health Department of Hebei Province Key Project and Leading Talent Project, China (grant no. zd2013087), the Tangshan City Science and Technology Project, China (grant no. 14130220B) and Youth Foundation of North China University of Science and Technology (grant no. z201736). References [1] H.C. Persson, L. Carlsson, K.S. Sunnerhagen, Life situation 5 years after subarachnoid haemorrhage, Acta Neurol. Scand. 137 (2018) 99e104. [2] H. Suzuki, F. Nakano, To improve translational research in subarachnoid hemorrhage, Transl. Stroke Res. 9 (2018) 1e3. [3] B.G. Lyeth, J.Y. Jiang, S.E. Robinson, H. Guo, L.W. Jenkins, Hypothermia blunts acetylcholine increase in CSF of traumatically brain injured rats, Mol. Chem. Neuropathol. 18 (1993) 247e256. lu, T. Deniz, Ü. Kisa, P. Atasoy, K. Aydinuraz, Effect of hypothermia on [4] O. Erog apoptosis in traumatic brain injury and hemorrhagic shock model, Injury 48 (2017) 2675e2682. [5] W. Choi, S.C. Kwon, W.J. Lee, Y.C. Weon, B. Choi, H. Lee, E.S. Park, R. Ahn, Feasibility and safety of mild therapeutic hypothermia in poor-grade subarachnoid hemorrhage: prospective pilot study, J. Kor. Med. Sci. 32 (2017) 1337e1344. [6] B. Levine, D.J. Klionsky, Development by self-digestion: molecular mechanisms and biological functions of autophagy, Dev. Cell 6 (2004) 463e477. [7] K. Maiese, Targeting molecules to medicine with mTOR, autophagy and neurodegenerative disorders, Br. J. Clin. Pharmacol. 82 (2016) 1245e1266. [8] S. Cao, S. Shrestha, J. Li, X. Yu, J. Chen, F. Yan, G. Ying, C. Gu, L. Wang, G. Chen, Melatonin-mediated mitophagy protects against early brain injury after subarachnoid hemorrhage through inhibition of NLRP3 inflammasome activation, Sci. Rep. 7 (2017) 2417. [9] X.Z. Li, C.Y. Sui, Q. Chen, X.P. Chen, H. Zhang, X.P. Zhou, Upregulation of cell surface estrogen receptor alpha is associated with the mitogen-activated protein kinase/extracellular signal-regulated kinase activity and promotes autophagy maturation, Int. J. Clin. Exp. Pathol. 8 (2015) 8832e8841. [10] Y. Chen, C. Luo, M. Zhao, Q. Li, R. Hu, J.H. Zhang, Z. Liu, H. Feng, Administration of a PTEN inhibitor BPV(pic) attenuates early brain injury via modulating AMPA receptor subunits after subarachnoid hemorrhage in rats, Neurosci. Lett. 588 (2015) 131e136. [11] J. Wu, Y. Zhang, P. Yang, B. Enkhjargal, A. Manaenko, J. Tang, W.J. Pearce, R. Hartman, A. Obenaus, G. Chen, J.H. Zhang, Recombinant osteopontin stabilizes smooth muscle cell phenotype via integrin receptor/integrin-linked kinase/rac-1 pathway after subarachnoid hemorrhage in rats, Stroke 47 (2016) 1319e1327. [12] W.S. Qiu, W.G. Liu, H. Shen, W.M. Wang, Z.L. Hang, Y. Zhang, S.J. Jiang, X.F. Yang, Therapeutic effect of mild hypothermia on severe traumatic head injury, Chin. J. Traumatol. 8 (2005) 27e32. [13] L. Huang, K. Huang, H. Ning, Autophagy induction by hispidulin provides protection against sevoflurane-induced neuronal apoptosis in aged rats, Biomed. Pharmacother. 98 (2017) 460e468. [14] F.J. Kong, J.H. Wu, S.Y. Sun, L.L. Ma, J.Q. Zhou, Liraglutide ameliorates cognitive decline by promoting autophagy via the AMP-activated protein kinase/ mammalian target of rapamycin pathway in a streptozotocin-induced mouse model of diabetes, Neuropharmacology 131 (2018) 316e325. [15] Y. Jin, Y. Lin, J.F. Feng, F. Jia, G.Y. Gao, J.Y. Jiang, Moderate hypothermia significantly decreases hippocampal cell death involving autophagy pathway after moderate traumatic brain injury, J. Neurotrauma 32 (2015) 1090e1100. [16] X. Xu, T. Zhi, H. Chao, K. Jiang, Y. Liu, Z. Bao, L. Fan, D. Wang, Z. Li, N. Liu, J. Ji, ERK1/2/mTOR/Stat3 pathway-mediated autophagy alleviates traumatic brain injury-induced acute lung injury, BBA-Biomembranes 1864 (2018) 1663e1674. [17] E.M. Halvorsen, J. Dennis, P. Keeney, T.W. Sturgill, J.B. Tuttle, J.B. Bennett Jr., Methylpyridinium, (MPP(þ))- and nerve growth factor-induced changes in pro- and anti-apoptotic signaling pathways in SH-SY5Y neuroblastoma cells, Brain Res. 952 (2002) 98e110. [18] S. Pattingre, C. Bauvy, P. Codogno, Amino acids interfere with the ERK1/2dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells, J. Biol. Chem. 278 (2003) 16667e16674.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Moderate hypothermia protects increased neuronal autophagy via activation of extracellular signal-regulated kinase signaling pathway in a rat model of early brain injury in subarachnoid hemorrhage Junjie Liu a, b, Wenji Liang a, Jingyao Wang a, Yaning Zhao a, b, Yichao Wang a, Jingxi Zhang a, Jianmin Li a, b, * a b
College of Clinical Medicine, North China University of Science and Technology, Tangshan, 063000, PR China Department of Neurosurgery, Affiliated Hospital of North China University of Science and Technology, Tangshan, 063000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2018 Received in revised form 21 May 2018 Accepted 23 May 2018 Available online 30 May 2018
Moderate hypothermia (MH) used as treatment for neurological diseases has a protective effect; however, its mechanism remains unclear. Neuronal autophagy is a fundamental pathological process of early brain injury in subarachnoid hemorrhage (SAH). We found that moderate activation of autophagy can reduce nerve cells damage. In this study, We found that MH can moderately increase the level of autophagy in nerve cells and improve the neurological function in rats. This type of autophagy activation is dependent on extracellular signal-regulated kinase (ERK) signaling pathways. The level of neuronal autophagy was down-regulated significantly by using U0126, an ERK signaling pathway inhibitor. In summary, these results suggest that MH can moderately activate neuronal autophagy through ERK signaling pathway, reduce nerve cell death, and produce neuroprotective effects. © 2018 Elsevier Inc. All rights reserved.
Keywords: Subarachnoid hemorrhage Autophagy Moderate hypothermia Neuroprotection Extracellular signal-regulated kinase
1. Introduction SAH is a type of cerebrovascular disease that is caused by intracranial vascular rupture due to various factors and seriously threatens human health. The cure rate for SAH has increased significantly with the advances in surgical techniques and medical equipment. However, its morbidity and mortality are still high. The surviving patients also have different degrees of cognitive decline, obstacles and dysfunctions; their quality of life is negatively affected; and their communities and families are bestowed a heavy burden [1]. SAH may induce early brain injury (EBI) and delayed brain injury, which can manifest as cerebral edema, intracranial hypertension, cerebral infarction, neurological disorders, and disturbances of consciousness. These symptoms may result from inflammation, cerebral vasospasm, apoptosis, and autophagy [2]. However, the current pathogenesis and treatment strategy for SAH remain poorly understood and thus warrant further study. Hypothermia at 28e35 C was defined as moderate hypothermia (MH) [3]. MH has made great breakthroughs in basic and * Corresponding author. College of Clinical Medicine, North China University of Science and Technology, Tangshan, 063000, PR China. E-mail address: [email protected] (J. Li). https://doi.org/10.1016/j.bbrc.2018.05.158 0006-291X/© 2018 Elsevier Inc. All rights reserved.
clinical research. MH may exert neuroprotective effects by reducing the markers of apoptotic pathway in TBI rats [4]. MH can reduce the risk of vasospasm and DCI in patients with SAH, improve functional outcomes, and reduce mortality [5]. However, only few studies focused on the effects and molecular mechanisms of MH in SAH. Autophagy is a key homeostatic process wherein cytosolic proteins and organelles are degraded and recycled. This process maintains cellular homeostasis and survival [6]. As a clearance pathway, autophagy exerts protective effects in multiple neurological disease models [7]. Autophagy can be activated in the SAH model, and this activation has neuroprotective effects [8]. However, the mechanism of autophagy activation is complex and involves multiple signal pathways. The up-regulation of cell surface estrogen receptor alpha is associated with mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) activity and promotes autophagy maturation in vitro [9]. This study aims to investigate the effects of MH in EBI after SAH in rats. In particular, we elucidate whether or not treatment with MH after SAH would protect rats against EBI and would moderately activate neuronal autophagy. Possible underlying mechanism(s) of any actions are also analyzed.
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2. Materials and methods
2.4. Manipulation of temperature
2.1. Animals
Frontal cortex brain temperature was monitored with a digital electronic thermometer (model DP 80; Omega Engineering, Stamford, CT) and a 0.15 mm-diameter temperature probe (model HYP033-1-T-G-60-SMP-M; Omega Engineering), which was inserted 4.0 mm ventral to the surface of the skull. The probe was removed before FPI and replaced immediately after injury. Rectal temperatures were measured with an electronic thermometer with analog display (model 43 TE; YSI, Yellow Springs, OH) and a temperature probe (series 400; YSI). The 32 C brain temperature was achieved by immersing the anesthetized rat body in ice-cold water. The skin and fur of all 21 animals were protected from water by placing the animal in a plastic bag (head exposed) before immersion. Animals were removed from the water bath when the brain temperature was reduced to within 2 C of the target temperature. Approximately 30 min was required to reach the target brain temperatures, which were maintained for 4 h under general anesthesia in room temperature through the intermittent application of ice packs as needed. A brain temperature of 37 C was achieved under general anesthesia with a heating blanket.
The experimental protocol was approved by the Animal Care and Use Committee of Central North China science and Technology University of China (No. 2013-99) and has conformed to the standards of the Guide for Care and Use of Laboratory Animals established by the National Institutes of Health of the United State of America. All efforts were taken to minimize the number of rats used and their suffering. Adult males Sprague Dawley (SD) rats that weigh 350e450 g and are 10e12 weeks old were supplied by Vital River Laboratories Ltd., Beijing, China. The license number was SCXK (Beijing): 2015-003. The animals were fed and given free access to water during experiments conducted in the Center of North China Science and Technology University and were allowed to adapt to feeding 14 days before the experiment. Feeding environment: temperature 18e26 C, relative humidity 40e70%, and temperature in the general rat feeding box must be 1e2 C higher than that in the environment and must have high humidity ~10%. Noise was below 85 dB, and ammonia concentration was below 20 PPm. Light and shade with alternating light were provided every day. 2.2. Experimental design 2.2.1. Experiment I Rats were randomly divided into SHAM, SAH model, and MH þ SAH group to determine the neuroprotective effect of MH treatment. SAH model and MH þ SAH group were further divided into four subgroups: 6, 24, 48, and 72 h. The neurological function of rats was detected by Garcia score table, The brain edema index was detected by dry-wet method and HE staining. The morphology of neurons in hippocampus CA1 area was also observed. 2.2.2. Experiment II Rats were randomly split into three groups: SHAM, SAH model, and MH þ SAH group to observe the changes on autophagy-related protein Beclin-1 and LC3 after MH treatment. Beclin-1 and LC3 in the hippocampus of SAH rats were detected 24 h after SAH by using IHC and Western blot. 2.2.3. Experiment III Rats were randomly divided into four groups: SAH þ vehicle (equal amount of DMSO solution), MH þ SAH, U0126 þ SAH dissolved in DMSO at a dose of 0.05 mg/kg (R & D systems, Minneapolis, MN, USA), and U0126 þ MH þ SAH (dissolved in DMSO at a dose of 0.05 mg/kg) to evaluate the role of the ERK signaling pathway in activating autophagy induced by MH. The neurological function of rats was detected by Garcia score table and the brain edema index was detected in the hippocampus by dry-wet method. The expression of phosphorylated ERK1/2 (p-ERK1/2) Beclin-1 and LC3 were detected in the hippocampus by Western blot. 2.3. Establishing the SAH model The SAH model was induced by endovascular perforation. While the mouse was under pentobarbital (50 mg/kg) anesthesia, the left carotid artery and its branches were exposed and separated. The left external carotid artery was cut, and a 4-0 monofilament suture was advanced into the internal carotid artery through it until resistance was felt. Then, the suture was inserted further to puncture the vessel and to induce SAH. The sham rats underwent the same procedure but without vessel puncture.
2.5. Neurological score Neurological score were evaluated by two blinded investigator [10], the animals from each group were tested for their neurological function by a modified Garcia Scale method for spontaneous activity, limb symmetry, forearm extension, climbing response, body sensation, and responsive touch. The scores for each part were added to obtain the total score of neurological function. According to the scale, a low score indicates severe nervous system injury. On the contrary, a high score indicates less nerve damage. The lowest score is 3 and the highest score is 18. 2.6. Water content in brain The brain was separated into four parts, including the left hemisphere, right hemisphere, cerebellum, and brain stem. Each part was immediately weighed (wet weight) and promptly dried for 72 h at 105 C (dry weight). The water content was then measured [11]. 2.7. HE staining of rats' brains Rats were subjected to deep anesthesia by 10% chloral hydrate. All rats were perfused transcardially by 4% paraformaldehyde in phosphate-buffered saline (PBS). The brain was subsequently harvested, and the brain tissue spanning from the optic chiasm to the cerebral transverse fissure was resected. The tissue was embedded in paraffin, cut into coronal sections (5 mm), and stained with hematoxylin and eosin. Sections were observed under an optical microscope. The sections were cut in a microtome and allowed to adhere to glass slides with polylysine. Images of the ipsilateral hippocampus were captured at 400 by using a microscope (Nikon Labophot; Nikon USA, Melville, NY). Specimens were examined by two pathologists (blinded to group conditions) to identify cell death based on characteristic cellular morphological changes. Eight rats were included in each of the four groups. 2.8. IHC analysis of rats' brains The immunoreactivity of LC3 and Beclin-1 was detected by subjecting the 4 l m-thick formalin-fixed, OCT-embedded sections to IHC analysis. Endogenous peroxidase was blocked with 3% H2O2
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for 5 min, followed by a brief rinse in distilled water and a 15 min wash in PBS. The sections were cooled at room temperature for 20 min and then rinsed in PBS. Nonspecific protein binding was blocked by incubation in 5% horse serum for 40 min. The sections were incubated with primary antibodies (Abs; anti-LC3 [CST3868], anti-beclin-1 [CST3738], anti-p-ERK1/2 [ab1776660] and anti-IgG [immunoglobulin G], all diluted 1:300; boaosen Biological Co, Beijing, China) for 1 h at room temperature, followed by a 15 min wash in PBS. After being washed three times with PBS, the sections were stained with 3,30 -diaminobenzidine (DAB), counterstained with hematoxylin, dehydrated, cleared, and sealed. For negative controls, the sections were incubated in the absence of a primary Ab. Ten microscopic fields per each section were photographed randomly for the counting of LC3 and Beclin-1-positive cells (400 magnifications, Nikon TE300; Nikon). An investigator blinded to group conditions had collected all nonbiased stereological data for the study.
3.3. MH treatment reduces neuronal injury in hippocampal CA1 area SAH
2.9. Western blot analysis of rats' brains
3.4. MH treatment increases neuronal autophagy
Frozen brain samples were mechanically lysed in 20 mM Tris (pH 7.6) containing 0.2% sodium dodecyl sulfate (SDS), 1% Triton X100, 1% deoxycholate, 1 mM of phenylmethylsulphonyl fluoride, and 0.11 IU/Ml aprotinin (Sigma-Aldrich, St. Louis, Mo). Lysates were centrifuged at 12,000 g for 20 min at 4 C. Protein concentration was estimated by Bradford's method. Samples (60 lg/lane) were separated by 12% SDS polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% skimmed milk for 1 h at room temperature and incubated with primary Abs against LC3 and Beclin-1 (1:1000 dilutes both from CST, Inc., Danvers, MA) at 4 C overnight. Glyceraldehyde-3phosphate dehydrogenase (diluted 1:10,000; Sigma-Aldrich) was used as loading control. After being washed three times in Trisbuffered saline (TBS)þTween-20 (TBST) for 10 min each, the membrane was incubated in the appropriate horseradish-peroxidaseeconjugated secondary Ab (diluted 1:10,000 in skimmed milk) for 2 h and washed three times in TBST for 10 min each. The enhanced chemiluminescence kit (GE Healthcare, United States) was applied to generate chemiluminescent signals. All western blotting data were analysis was performed using ImageJ 6.0 software (National Institutes of Health, Bethesda, MD, United States).
Beclin-1 IHC staining was brown and was mainly expressed in the neuronal cell plasma. The Sham operation group showed occasional immune-positive cells and was lightly stained. Nerve cells were neatly arranged and had normal structure and nucleolus. The expression of SAH þ MH was significantly increased in the SAH group than in the Sham group. The former group was stained darker with many immunopositive cells. LC3 IHC staining was brown and was mainly expressed in the neuronal cell plasma. The Sham group showed occasional immune-positive cells and light staining. Its neurons were arranged in neat rows, had normal structure and clear nucleoli. Compared with that in the Sham group, immunity is significantly enhanced in the SAH group. Compared with the SAH group, SAH þ MH group was stained darker and had more immunopositive cells (Fig. 2A and B). Western blotting was performed in sham operation, SAH model, and HM þ SAH group after euthanizing the rats. Beclin-1 was detected at 24 h after SAH, and its expression in HM þ SAH group was significantly higher than that in SAH model group (1.25 ± 0.43 vs.1.48 ± 0.38, P < 0.05). LC3 was also detected at 24 h after SAH, and its expression in HM þ SAH group was significantly higher than that in SAH model group (1.34 ± 0.29 vs.1.61 ± 0.41, P < 0.05) (Fig. 2C, D, and 2E).
3. Results
3.5. Effect of inhibitor of ERK signaling on neurological function and cerebral edema index at 24 h in SAH group
3.1. MH treatment improves neurological function after SAH Fig. 1A and B shows that the sham operation group and the model group were successfully established. Compared with the sham operation group (17.8 ± 1.0), SAH group showed neurological impairment at each time point. Compared with those in the SAH model group, the neurological deficits in the MH þ SAH treated group were significantly relieved at all the time points and reached the highest level at 72 h (10.01 vs.14.85,P < 0.05) (Fig. 1C). 3.2. MH treatment reduces brain edema after SAH Compared with the sham operation group (78.3 ± 0.3), SAH group showed increased brain edema at each time point. Compared with those in the SAH model group, brain edema deficits in the MH þ SAH treated group were significantly relieved at all the time points, and the highest level (82.7 ± 0.4 vs.81.4 ± 0.1, P < 0.05) was observed at 24 h (Fig. 1D).
The neurons in the hippocampus CA1 area of Sham group were arranged neatly. The nucleus was positive, the nucleolus was clearly visible, the inclusion pulp was indifferent, and the axons were evenly seen. In the SAH group, the neurons were arranged relatively loosely. The hierarchical structure was disrupted, and the structure of the tissue was severely damaged. Many nucleus pyknosis and nucleolus disappeared. The surrounding tissue dissolved and thus showed the vascular components. Compared with the SAH group, MH þ SAH group showed significantly reduced neuronal damage, relatively mitigated cell edema, relatively neat structure, and less neuron death (Fig. 1E). Statistical analysis shows that the neuronal mortality in MH þ SAH group was significantly lower than that in SAH group and reach the lowest level (40.79% ± 1.9% vs.29.15% ± 3.6%, P < 0.05) at 6 h (Fig. 1F).
Compared with the Vehicle þ SAH group, MH þ SAH group showed neurological impairment (4.12 ± 1.3 vs. 6.57 ± 1.2, P < 0.05). Compared with that in the MH þ SAH group, the neurological impairment in U0126 þ SAH group was significantly reduced (6.57 ± 1.2 vs. 4.15 ± 0.9, P < 0.05). No significant difference in neurological score was detected between U0126 þ MH þ SAH and U0126 þ SAH groups (Fig. 3A). Compared with the Vehicle þ SAH group, MH þ SAH group showed reduced brain edema (82.50 ± 0.5 vs. 79.44 ± 1.2, P < 0.05). Compared with that in the MH þ SAH group, brain edema in the U0126 þ SAH group was increased significantly (79.44 ± 1.2 vs. 82.49 ± 0.8, P < 0.05). No significant difference in brain edema was detected between U0126 þ MH þ SAH and U0126 þ SAH groups (Fig. 3B). 3.6. Effects of ERK signaling pathway inhibitors on neuronal autophagy after 24 h in SAH group U0126 treatment significantly inhibited the expression of p-
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Fig. 1. Neuroprotective effect of moderate hypothermia (MH) on early brain injury (EBI) in rats with subarachnoid hemorrhage (SAH). (A) Bulk specimen sham of operation and model group; (B) IHC analysis shows of the sham operation and the model group under the microscope (200 ); (C) Garcia score of SHAM, SAH, and MH þ SAH group, at 6, 24, 48, and 72 h; (D) Brain water content in MH þ SAH and SAH group, at 6, 24, 48, and 72 h; (E) Damage on nerve cells in SHAM, SAH, and MH þ SAH groups (HE, 400 ); (F) Neuronal cell death rate in SHAM, SAH, and MH þ SAH groups was significantly lower than that in SAH group at 6, 24, 48, and 72 h. According to t-test, *P˂0.05, **P˂0.01. Values are mean ± SD (n ¼ 6 independent experiments).
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Fig. 2. Effect of MH on the expression of autophagy-related proteins Beclin-1 and LC3 in SAH rats. (A) IHC images of the localization and expression of Beclin-1 and LC3 in hippocampus CA1; (B) Results of quantitative analysis of IHC; (C) Western blot analysis of the Beclin-1 and LC3 expression in hippocampus at 24 h after SAH; (D) Quantification analysis of Beclin-1 expression in hippocampus at 24 h in SAH group; (E) Quantification analysis of LC3 expression in hippocampus at 24 h after SAH. According to t-test, ***p < 0.01, ***p < 0.001 vs. SHAM; ##p < 0.01 vs. SAH, Values are mean ± SD, (n ¼ 6 independent experiments).
ERK1/2, which was significantly down-regulated in U0126 þ SAH group compared with that in vehicle þ SAH group (0.65 ± 0.10 vs.0.25 ± 0.09, P < 0.0001). Compared with that in the Vehicle þ SAH group, the expression of ERK1/2 was significantly up-regulated in MH þ SAH group (0.65 ± 0.10 vs. 0.81 ± 0.08, P < 0.001). ERK1/2 expression was not significantly different between U0126 þ MH þ SAH and MH þ SAH groups (Fig. 3C and D). U0126 treatment significantly inhibited the expression of Beclin-1, which was significantly down-regulated in U0126 þ SAH group compared with that in vehicle þ SAH group (0.54 ± 0.11 vs.0.19 ± 0.09, P < 0.0001). Beclin-1 expression was not significantly different between U0126 þ MH þ SAH and MH þ SAH groups (Fig. 3C and E). U0126 treatment significantly inhibited the transformation from LC3-I to LC3-II. The expression of LC3-Ⅱ was significantly
down-regulated in U0126 þ SAH group compared that in vehicle þ SAH group (0.43 ± 0.09 vs. 0.18 ± 0.07, P < 0.001). Compared with that in Vehicle þ SAH group, the expression of LC3II in MH þ SAH group was significantly increased (0.43 ± 0.09 vs. 0.67 ± 0.12, P < 0.001). LC3-II expression was not significantly different in U0126 þ SAH and U0126 þ MH þ SAH groups (Fig. 3C and F). 4. Discussion In this study, we found that SAH causes hippocampal cell death, decreases neurological function, and increases mouse cerebral edema, which further peaked at 24 h post-SAH. MH could attenuate hippocampal cell death, reduce brain edema, and improve neurological function. We also found that autophagy increased after SAH
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Fig. 3. HM-affected autophagy is dependent on the ERK signaling pathway after SAH. (A) Neuroprotective effect of MH after U0126 treatment; (B) U0126 can aggravate the degree of brain edema after moderate hypothermia; (C) Western-blot analysis of the p-ERK1/ 2,Beclin-1 and LC3 expression in hippocampus at 24 h after SAH; (D) Quantification analysis of p-ERK1/2 expression in hippocampus at 24 h after SAH; (E) Quantification analysis of Beclin-1 expression in hippocampus at 24 h after SAH; (F) Quantification analysis of LC3 expression in hippocampus at 24 h after SAH. According to t-test, *p < 0.05, ***p < 0.001 vs. Vehicle, ###p < 0.05, ###p < 0.001 vs. MH, Values are mean ± SD (n ¼ 6 independent experiments).
and further increased after MH treatment. The results suggest that the autophagy moderate activation at EBI of SAH by MH may be a neuroprotective mechanism. However, the relationship of MH with autophagy is unknown. We detected the sensitive indicator of ERK signaling pathway P-ERK1/2 and found that the autophagy activation by MH is dependent on the ERK signaling pathway. MH refers to light and moderate low temperatures of 30e34 C. It was found that MH can significantly improve the prognosis of patients with brain injury [12]. MH is a feasible treatment and can
be safely used in patients with poor-grade SAH. In addition, this process may reduce the risk of vasospasm and DCI, thus improving the functional outcomes and reducing mortality [5]. However, the neuroprotective effect of MH on SAH is still poorly understood. Our study show that the neuroprotective effects of MH have been validated in the rat model of disease, MH treatment can significantly reduce neuronal death in the hippocampus of SAH rats and improve the neurological function scores. Therefore, we believe that MH has a neuroprotective effect on SAH.
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Induced Moderate autophagy activation is essential to maintain cellular homeostasis and cell survival. Experimental data show that autophagy has neuroprotective effect. Hispidulin significantly induces autophagy in H4 cells. This phenomenon contributes to its protective activity against sevoflurane-induced apoptosis [13]. These findings highlight that hispidulin offers neuroprotection against sevoflurane-induced cognitive dysfunction, which is mediated by autophagy. Liraglutide can promote autophagy, thereby reducing neuronal damage in the hippocampus and promoting cognitive recovery in diabetic cognitive disorder mice. Autophagy is known to have neuroprotective effects [14]. MH can increase autophagy in the hippocampal neurons and thus reduce brain damage in the TBI rat model [15]. However, whether or not the neuroprotective effect of MH on SAH can also increase the autophagy of the nerve cells remain unclear. Beclin-1 and LC3 are biomarkers for detecting changes in autophagy. We found that Beclin-1 and LC3 expression were up-regulated after MH treatment in rats. Therefore, we believe that the neuroprotective effect of MH on SAH rats is achieved by up-regulating the autophagy of nerve cells. ERK plays an important role in regulating autophagy. The ERK signal cascade is activated in several well established autophagy models, and inhibition of this pathway inhibits autophagy [16]. ERK signaling pathway inhibitor U0126 induces the expression of neurotoxin 1-methyl-4-phenylpyridinium (MPP) in damaged neurons, reduces the level of autophagy protein LC3 cells, and increases mitochondrial damage [17]. ERK1/2 activators stimulate the phosphorylation of Galpha-interacting protein (GAIP) to stimulate macrophages in human colon cancer cell line HT-29 [18]. Our experimental results showed that the expression of Beclin-1 and LC3 in the hippocampus of rats was significantly down-regulated. The damage of nerve cells was intensified after the U0126, ERK signaling pathway inhibitor was applied. The activation of autophagy was regulated by MEK/ERK signaling pathway in EBI of SAH. As a sensitive indicator of ERK signaling pathway, p-ERK1/2 was detected in this experiment. We found that the effect of MH on autophagy depends on the ERK signaling pathway. In this study, we observed that SAH could increase autophagy in the hippocampus, and MH could accelerate this process. Autophagy is the main mechanism for the bulk elimination of aberrant cell components and could interact with other cell death mechanisms. We found that MH could attenuate cell death in the ipsilateral hippocampus. Furthermore, this process relies on the ERK signaling pathway. Therefore, we propose that the neuroprotective effects of MH may be related to the interaction between autophagy and ERK signaling pathway. In conclusion, autophagy pathway as mediated by ERK signaling pathway may participate in the neuroprotective effect of post-SAH MH. However, further work will be required to reveal the possible upstream target molecules of the promotion of autophagy via ERK signaling pathway by MH. Author contribution Jianmin Li designed research; junjie Liu, yaning Zhao, Jingxi Zhang and Jingyao Wang performed research; Yichao Wang analyzed data; Wenji Liang wrote the paper. All authors reviewed
the manuscript. Acknowledgements This work was supported by the Health Department of Hebei Province Key Project and Leading Talent Project, China (grant no. zd2013087), the Tangshan City Science and Technology Project, China (grant no. 14130220B) and Youth Foundation of North China University of Science and Technology (grant no. z201736). References [1] H.C. Persson, L. Carlsson, K.S. Sunnerhagen, Life situation 5 years after subarachnoid haemorrhage, Acta Neurol. Scand. 137 (2018) 99e104. [2] H. Suzuki, F. Nakano, To improve translational research in subarachnoid hemorrhage, Transl. Stroke Res. 9 (2018) 1e3. [3] B.G. Lyeth, J.Y. Jiang, S.E. Robinson, H. Guo, L.W. Jenkins, Hypothermia blunts acetylcholine increase in CSF of traumatically brain injured rats, Mol. Chem. Neuropathol. 18 (1993) 247e256. lu, T. Deniz, Ü. Kisa, P. Atasoy, K. Aydinuraz, Effect of hypothermia on [4] O. Erog apoptosis in traumatic brain injury and hemorrhagic shock model, Injury 48 (2017) 2675e2682. [5] W. Choi, S.C. Kwon, W.J. Lee, Y.C. Weon, B. Choi, H. Lee, E.S. Park, R. Ahn, Feasibility and safety of mild therapeutic hypothermia in poor-grade subarachnoid hemorrhage: prospective pilot study, J. Kor. Med. Sci. 32 (2017) 1337e1344. [6] B. Levine, D.J. Klionsky, Development by self-digestion: molecular mechanisms and biological functions of autophagy, Dev. Cell 6 (2004) 463e477. [7] K. Maiese, Targeting molecules to medicine with mTOR, autophagy and neurodegenerative disorders, Br. J. Clin. Pharmacol. 82 (2016) 1245e1266. [8] S. Cao, S. Shrestha, J. Li, X. Yu, J. Chen, F. Yan, G. Ying, C. Gu, L. Wang, G. Chen, Melatonin-mediated mitophagy protects against early brain injury after subarachnoid hemorrhage through inhibition of NLRP3 inflammasome activation, Sci. Rep. 7 (2017) 2417. [9] X.Z. Li, C.Y. Sui, Q. Chen, X.P. Chen, H. Zhang, X.P. Zhou, Upregulation of cell surface estrogen receptor alpha is associated with the mitogen-activated protein kinase/extracellular signal-regulated kinase activity and promotes autophagy maturation, Int. J. Clin. Exp. Pathol. 8 (2015) 8832e8841. [10] Y. Chen, C. Luo, M. Zhao, Q. Li, R. Hu, J.H. Zhang, Z. Liu, H. Feng, Administration of a PTEN inhibitor BPV(pic) attenuates early brain injury via modulating AMPA receptor subunits after subarachnoid hemorrhage in rats, Neurosci. Lett. 588 (2015) 131e136. [11] J. Wu, Y. Zhang, P. Yang, B. Enkhjargal, A. Manaenko, J. Tang, W.J. Pearce, R. Hartman, A. Obenaus, G. Chen, J.H. Zhang, Recombinant osteopontin stabilizes smooth muscle cell phenotype via integrin receptor/integrin-linked kinase/rac-1 pathway after subarachnoid hemorrhage in rats, Stroke 47 (2016) 1319e1327. [12] W.S. Qiu, W.G. Liu, H. Shen, W.M. Wang, Z.L. Hang, Y. Zhang, S.J. Jiang, X.F. Yang, Therapeutic effect of mild hypothermia on severe traumatic head injury, Chin. J. Traumatol. 8 (2005) 27e32. [13] L. Huang, K. Huang, H. Ning, Autophagy induction by hispidulin provides protection against sevoflurane-induced neuronal apoptosis in aged rats, Biomed. Pharmacother. 98 (2017) 460e468. [14] F.J. Kong, J.H. Wu, S.Y. Sun, L.L. Ma, J.Q. Zhou, Liraglutide ameliorates cognitive decline by promoting autophagy via the AMP-activated protein kinase/ mammalian target of rapamycin pathway in a streptozotocin-induced mouse model of diabetes, Neuropharmacology 131 (2018) 316e325. [15] Y. Jin, Y. Lin, J.F. Feng, F. Jia, G.Y. Gao, J.Y. Jiang, Moderate hypothermia significantly decreases hippocampal cell death involving autophagy pathway after moderate traumatic brain injury, J. Neurotrauma 32 (2015) 1090e1100. [16] X. Xu, T. Zhi, H. Chao, K. Jiang, Y. Liu, Z. Bao, L. Fan, D. Wang, Z. Li, N. Liu, J. Ji, ERK1/2/mTOR/Stat3 pathway-mediated autophagy alleviates traumatic brain injury-induced acute lung injury, BBA-Biomembranes 1864 (2018) 1663e1674. [17] E.M. Halvorsen, J. Dennis, P. Keeney, T.W. Sturgill, J.B. Tuttle, J.B. Bennett Jr., Methylpyridinium, (MPP(þ))- and nerve growth factor-induced changes in pro- and anti-apoptotic signaling pathways in SH-SY5Y neuroblastoma cells, Brain Res. 952 (2002) 98e110. [18] S. Pattingre, C. Bauvy, P. Codogno, Amino acids interfere with the ERK1/2dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells, J. Biol. Chem. 278 (2003) 16667e16674.