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Remote limb ischemic postconditioning protects against cerebral ischemia-reperfusion injury by activating AMPK-dependent autophagy
Accepted Manuscript Title: Remote limb ischemic postconditioning protects against cerebral ischemia-reperfusion injury by activating AMPK-dependent autophagy Authors: Hao Guo, Lei Zhao, Bodong Wang, Xia Li, Hao Bai, Haixiao Liu, Liang Yue, Wei Guo, Zhenyuan Bian, Li Gao, Dayun Feng, Yan Qu PII: DOI: Reference:
S0361-9230(17)30605-6 https://doi.org/10.1016/j.brainresbull.2018.02.013 BRB 9380
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
16-10-2017 26-1-2018 9-2-2018
Please cite this article as: Hao Guo, Lei Zhao, Bodong Wang, Xia Li, Hao Bai, Haixiao Liu, Liang Yue, Wei Guo, Zhenyuan Bian, Li Gao, Dayun Feng, Yan Qu, Remote limb ischemic postconditioning protects against cerebral ischemiareperfusion injury by activating AMPK-dependent autophagy, Brain Research Bulletin https://doi.org/10.1016/j.brainresbull.2018.02.013 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.
Remote limb ischemic postconditioning protects against cerebral ischemia-reperfusion injury by activating AMPKdependent autophagy
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Hao Guo a, 1, Lei Zhao a, 1, Bodong Wang a, b, 1, Xia Li a, Hao Bai a, Haixiao Liu a, Liang Yue a, c, Wei Guo a, Zhenyuan Bian d, Li Gao a, Dayun Feng a,
a
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Yan Qu a, *
Department of Neurosurgery, Tangdu Hospital, The Fourth Military
Department of Neurosurgery, Jinan Military General Hospital, Jinan
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b
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Medical University, Xi’an 710038, China
Department of Neurosurgery, Xi’an Aerospace General Hospital, Xi’an
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c
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250031, China
d
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710100, China
Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth
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Military Medical University, Xi’an 710032, China
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These authors contribute equally to this work.
*
Corresponding author: Yan Qu, Department of Neurosurgery, Tangdu
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Hospital, Fourth Military Medical University, No. 569 Xinsi Road, Xi’an 710038, China. E-mail: [email protected].
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Highlights: RIPoC markedly decreases neuronal injury and infarct size after cerebral I/R.
RIPoC alleviates neuronal apoptosis after cerebral I/R.
RIPoC activates AMPK-dependent autophagy after cerebral I/R.
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Abstract
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Remote limb ischemic postconditioning (RIPoC) is a promising adjunct treatment for cerebral ischemia-reperfusion (IR) injury. However, the
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underlying mechanisms have not been fully elucidated yet. The present
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study aims to investigate potential involvement and regulatory mechanisms
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of autophagy in RIPoC treatment against cerebral IR injury in mice. Mice
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were subjected to 2 h middle cerebral artery occlusion (MCAO) then
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treated with vehicle, 3-methyladenine (3-MA, an autophagy inhibitor), or compound C (an AMPK inhibitor) at the onset of reperfusion. RIPoC was
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carried out by 3 cycles of 10-min occlusion-reperfusion of bilateral femoral
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artery at the beginning of the reperfusion. Infarct volume, neurological score, and brain water content of the mice were assessed after 12 h reperfusion. Autophagy markers, cell apoptosis markers, and AMPK
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pathway activity were also evaluated. Our results indicated that RIPoC treatment reduced neurological deficits, brain water content, and infarct volume after IR. Meanwhile, RIPoC was proved to induce autophagy and activate AMPK pathway. Furthermore, the RIPoC-induced autophagy and 2
neuroprotection were abolished by 3-MA and partially blocked by compound C. In conclusion, the present study suggests that RIPoC attenuates cerebral IR injury by activating AMPK-dependent autophagy.
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Key words: remote limb ischemic postconditioning, cerebral ischemia-
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reperfusion injury, AMPK, autophagy
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Introduction
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Stroke is one of the most common diseases with a leading cause of high
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morbidity and mortality worldwide [28]. Over 80% of all strokes are
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ischemic which were caused by insufficient blood supply to the brain.
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Novel therapeutic methods and medical devices have decreased the mortality and disability rate of acute ischemic stroke (AIS) over the last
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decades [2]. Although Solitaire stent retriever and alteplase (rtPA) have
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been approved by the FDA for the treatment of AIS, the outcome remains unsatisfactory due to the narrow time window, side effects, and some other factors [14]. Besides, despite the success of reperfusion therapy by
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endovascular treatment and thrombolysis, the ischemia-reperfusion (IR) injury is still of significant concern [9]. Therefore, adjunct therapies on top of timely and effective reperfusion are still needed to improve the clinical outcome and reduce the infarct size of patients with AIS. 3
Ischemic conditioning (IC) encompasses a number of related endogenous neuroprotective strategies which conduct a series of transient periods of mechanical occlusions and reperfusions to protect against vital organ ischemia [15]. It can be applied either directly to the target organ or
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from afar, for example limbs. Remote limb ischemic postconditioning (RIPoC) is an efficacious protective strategy which performs ischemic
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condition on a remote organ (limbs) after the ischemic period of the brain
to protect the brain against IR injury in experimental stroke. It has attracted
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extensive interest recently. Although the neuroprotection of RIPoC has
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been successfully proved in several studies, the underlying mechanisms
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have not been fully elucidated yet [3, 25].
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Autophagy is an evolutionarily conserved process in which
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unnecessary or dysfunctional proteins and organelles are delivered to the lysosomes for degradation [24]. As a vital degradation/recirculation system
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in at the cellular level, autophagy is considered to play an important role in
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many pathological conditions, including AIS [37]. Although it is undisputable that autophagy can be activated after cerebral ischemia, whether autophagy plays a protective or detrimental role in IR injury
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remains controversial [31]. AMP-activated protein kinase (AMPK) is a highly conserved crucial energy sensor which regulates cellular metabolism to maintain energy homeostasis [8]. AMPK signaling pathway also regulates autophagy as previously demonstrated [16]. In particularly, 4
AMPK has been proved to phosphorylate and activate unc-51-like autophagy-activating kinase 1 (ULK1), which triggers the initiation of the autophagic cascade [5]. In this investigation, we aim to explore the potential involvement and function of autophagy in RIPoC treatment
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of AMPK-dependent autophagy in RIPoC-induced protection.
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against cerebral IR injury in mice. Moreover, we also investigate the role
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Materials and methods
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Reagents
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Dimethyl sulfoxide (DMSO), 2,3,5-triphenyltetrazolium chloride
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(TTC), and 4’,6-diamino-2-phenylindole (DAPI) were purchased from
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Sigma-Aldrich (St. Louis, MO, USA). Bicinchoninic acid (BCA) protein assay kits were purchased from Beyotime Biotech Co. (Shanghai, China).
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3-Methyladenine (3-MA) was purchased from Selleckchem (Houston, TX,
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USA). Compound C (CC) (ab146597) and Rabbit polyclonal antibody against active caspase-3 (ab2302) was obtained from Abcam (Cambridge, UK). Rabbit monoclonal antibodies against Bcl-2 (#2870)，Bax (#14796) ,
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β-actin (#4970), LC3B (#3868), Beclin-1 (#3495), Atg7 (#8558), SQSTM1/P62 (#8025), AMPKα (#5831), Phosphor-AMPKα (Thr172) (#50081), mTOR (#2983), Phosphor-mTOR (Ser2448) (#5536), Acetyl-CoA Carboxylase (ACC) (#3676), Phosphor-ACC (Ser79) (#11818), unc-51-like 5
autophagy-activating kinase 1 (ULK1) (#8054), and Phosphor-ULK1 (Ser555) (#5869) were purchased from Cell Signaling Technology (Beverly, MA, USA). The HRP-conjugated rabbit anti-mouse secondary antibody and HRP-conjugated goat anti-rabbit secondary antibody were purchased
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from Bioworld Co. (Shanghai, China). Terminal deoxynucleotidyl transferased UTP nick-end labeling (TUNEL) kit was purchased from
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Roche (Mannheim, Germany).
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Experimental animals
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All animals used in this study received humane care according to the
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Guide for the Care and Use of Laboratory Animals, which was published
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by the US National Institutes of Health (National Institutes of Health
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Publication no. 85-23, revised 1996). All protocols were approved by the Ethics Committee of the Fourth Military Medical University.
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Healthy adult male C57BL/6 J mice weighing 20-25g were purchased
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from the Experimental Animal Center of the Fourth Military Medical University. The mice were kept in a pathogen-free environment at a constant temperature (23 °C) and humidity (60 %), with a 12-h light/dark
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cycle and had free access to food and water for two weeks before the experiment as an acclimatization period.
Induction of MCAO 6
Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) as described previously [35]. Briefly, mice were anesthetized with a mixture of isoflurane (3 % induction, 1.5 % maintenance, v/v) in 30 % oxygen and 70 % nitrous oxide. Heating lamps
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were used to maintain temperature at 36.5-37 °C. A midline neck incision was prepared. Then, the right common carotid artery (CCA), internal
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carotid artery (ICA) and external carotid artery (ECA) were exposed and isolated under an operating microscope. After the CCA was ligated close
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to its origin with a 3-0 silk suture, a 6-0 nylon monofilament suture (Beijing
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Sunbio Biotech Co. Ltd, Beijing, China), blunted and coated with a
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rounded tip, was inserted through a small incision into the CCA and
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advanced 10 mm distal to the carotid bifurcation until mild resistance to
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occlude the origin of the MCA. Reperfusion was allowed after 2 h of MCAO by monofilament removal. Sham-operated animals received
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not cut.
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midline neck incisions. The right common carotid artery was isolated, but
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Induction of RIPoC RIPoC was induced using an open invasive technique after 2 h of
MCAO. Under anesthesia, right femoral artery was exposed. RIPoC was induced by three cycles of 10-min of bilateral femoral arteries occlusion by a microvascular clamp to occlude the femoral vessels under an operating 7
microscope, followed by 10-min of reperfusion. Bilateral distal limb pallor were observed during occlusion, followed rapidly by brisk reactive hyperemia during reperfusion. Anesthesia was discontinued and animals were placed back into their cages. In the sham and IR model groups, the
Drug administration Intracerebroventricular
(ICV)
injection
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femoral arteries were only exposed and isolated.
was
performed
under
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isoflurane anesthesia. Mice were placed on a stereotaxic apparatus with the
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skull held horizontally. 3-MA and CC were dissolved in DMSO separately.
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Mice were administered an ICV injection of vehicle or 3-MA or CC at the
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onset of reperfusion. Control mice were injected with the same volume of
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DMSO. Vehicle, 3-MA, or CC was infused 0.5 μL/min into lateral ventricle using a 10 µL Hamilton syringe at the following coordinate: 0.46 mm
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rostral, 1mm lateral in relation to bregma, and 2.8 mm depth from the
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surface. The needle was kept in place for 10 min after injection completion and then withdrawn slowly.
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Experimental design Mice were randomized divided into the following groups (Fig. 1). 1. Sham group, in which mice were subjected to the same as surgical operation, but without occlusion of the right middle cerebral artery and 8
RIPoC treatment. 2. IR group, in which mice were subjected to 2 h of MCAO followed by reperfusion. 3. IR + RIPoC group, in which mice received bilateral hindlimb
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intervention with 3 cycles of 10-min occlusion-reperfusion immediately at the onset of reperfusion.
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4. IR + Veh group, in which mice were administered vehicle by ICV injection at the onset of reperfusion.
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5. IR + Veh + RIPoC group, in which mice were administered vehicle by
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ICV injection and bilateral hindlimb intervention with 3 cycles of 10-min
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occlusion-reperfusion at the onset of reperfusion.
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6. IR + 3-MA group, in which mice were administered 3-MA by ICV
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injection at the onset of reperfusion.
7. IR + 3-MA + RIPoC group, in which mice were administered 3-MA by
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ICV injection and bilateral hindlimb intervention with 3 cycles of 10-min
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occlusion-reperfusion at the onset of reperfusion. 8. IR + CC group，in which mice were administered CC by ICV at the onset of reperfusion.
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9. IR+ CC + RIPoC group, in which mice were administered CC by ICV injection and bilateral hindlimb intervention with 3 cycles of 10-min occlusion-reperfusion at the onset of reperfusion.
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Measurement of neurological deficit and cerebral edema The mice were returned to their cages after the procedures were finished and again allowed free access to food and water. The neurological deficit score (NDS) was assessed just before sacrifice at 12 h after
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reperfusion as described previously [35]. Each mouse was scored by three examiners who were blinded to the identity of the mice or treatment
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protocol. NDS were evaluated as follows: 0, no deficit; 1, forelimb weakness and torso turning to the ipsilateral side when held by tail; 2,
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circling to the contralateral side but normal posture at rest; 3, unable to bear
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weight on the affected side at rest and 4, no spontaneous motor activity or
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barrel rolling. If no deficit was observed 2 h after the beginning of the
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occlusion period, the animal was removed from further study.
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The water content of brain was assessed at 12 h after reperfusion. As described previously [35], mice were decapitated under deep anesthesia.
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The brains were quickly removed and the hemispheres were separated in
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the sagittal plane. After the wet weight of the brain tissues was quantified, the tissues were desiccated at 105°C for 48 h until the weight was constant (dry weight). The water content of each brain was calculated as follow:
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(wet weight - dried weight) /wet weight × 100 %.
Measurement of infarct volume After a reperfusion period of 12 h, the cerebral infarct area was 10
identified by TTC staining as described previously [35]. The mice were sacrificed by rapid decapitation under deep anesthesia. The whole brain was rapidly removed and cut into 2-mm-thick coronal sections, then the slices were stained with 2 % solution of TTC diluted in 0.1 M sodium
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phosphate buffered saline (PBS) (pH 7.4) at 37 °C for 30-min in the dark and immersed 30-min in 10 % formalin. The infarct volume was calculated
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according to the following formula: V = t × (A1 + A2... + An), in which ‘t’ is the brain slice thickness and ‘A’ is the infarct area. The percentage
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cerebral infarct volume was measured as follows: (cerebral infarct total
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volume/whole brain volume) ×100 %.
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Western blot
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Western blot was performed as described previously [20]. After a reperfusion period of 12 h, mice were sacrificed under deep anesthesia and
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perfused with saline solution. The cerebral cortex from the right
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hemisphere of each mouse was homogenized and the total proteins were extracted by RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentrations were determined by BCA protein assay kit. Equal amounts
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of protein (40μg) were separated by 10-15% sodium dodecyl sulfatepolyacrylamide gelelectrophoresis (SDS-PAGE) gels electrophoresis and then transferred to PVDF membranes. The membranes were blocked by 2% bull serum albumin (BSA) in Tris-buffered saline and Tween 20 (pH 7.6) 11
(TBST) for 1 h at room temperature and were then incubated overnight at 4 °C with antibodies against Bax, Bcl-2, active caspase-3, LC3B, Beclin1, Atg7, SQSTM1/P62, AMPK, p-AMPK, mTOR, p-mTOR, ACC, p-ACC, ULK 1, p-ULK 1, and β-actin at 4°C overnight. The membranes were then
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incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:20,000 dilution) at room temperature for 2 h and washed with
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TBST. The protein bands were detected using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA) and quantified using the Quantity One
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software package (West Berkeley, CA, USA).
apoptosis
in the tissue was detected
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Cell
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TUNEL staining
using terminal
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deoxynucleotidyl transferased UTP nick end labeling (TUNEL) staining 12 h after cerebral IR induction in accordance with the manufacturer's
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instructions [18]. The samples were treated with 0.3% hydrogen peroxide
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for 30 minutes and incubated with 0.25% proteinase K for 45 minutes at 37ᵒC. Then, the slices were dyed using TUNEL reaction solution prepared according to the manufacturer instructions in a humidified dark chamber
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for 1 h at 37ᵒC. Finally, the tissues were dyed using DAPI for 10 minutes at 37ᵒC. Meanwhile, each of these steps was followed by three 5-minute PBS washes. Sections were covered using cover glasses with anti-fade mounting medium. The TUNEL-positive cells showed green fluorescence 12
and nucleus were stained with blue fluorescence. Apoptotic index was determined as the ratio of the number of TUNEL-positive neurons to the total number of neurons. Five visual fields were randomly chosen from the ischemic penumbra of each slice under a high-power lens for counting
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apoptotic cells.
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Immunofluorescence
The sections were stained with rabbit monoclonal antibody against
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LC3B (1:200) at 4 ℃ , followed by goat anti-rabbit FITC-conjugated
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conjugated secondary antibody (CW0114, ComWin Biotech Co., Beijing,
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China) for 2 hours at room temperature. Then, the sections were incubated
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with DAPI staining solution for 10 min at room temperature. Photographs
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were snapped with a confocal fluorescent microscope (Nikon Eclipse Ti).
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Statistical analysis
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GraphPad Prism 6 was used to analyze data in this study. Data were presented as the mean ± SD. NDSs were analyzed by the non-parametric Kruskal-Wallis test. Other data were tested using one-way ANOVA. Tukey
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post hoc analysis was used for intergroup comparisons. P<0.05 was considered statistically significant.
Results 13
RIPoC protected against cerebral IR injury
To determine whether RIPoC could attenuate IR injury in ischemic brain,
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mice were subjected to 10 minutes of ischemia followed by 10 minutes of reperfusion on bilateral hindlimb immediately at the onset of cerebral
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reperfusion. NDS, infarct volumes, and cerebral edema were assessed 12 h after the IR insult. Based on these measures, NDS, brain water content, and
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infarct volumes increased dramatically after MCAO. RIPoC treatment
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significantly reduced NDS, brain water content, and infarct volume.
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Similar results were obtained for TUNEL staining (Fig. 2). The levels of
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Bax, active caspase-3, and Bcl-2 were measured using western blot. As
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shown in Fig. 2, the levels of Bax and active caspase-3 were significantly lower in the IR + RIPoC group than those levels in the IR group. By
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contrast, the RIPoC dramatically increased Bcl-2 expression level in the IR
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+ RIPoC group compared with the IR group (Fig. 2).
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RIPoC induced autophagy during cerebral IR injury
To determine the effect of RIPoC on autophagy in cerebral IR process, the levels of the autophagy markers LC3B, Beclin-1, Atg7, and SQSTM1/ P62 were detected. We found that RIPoC significantly increased the levels of 14
LC3B-II/LC3B-I, Beclin-1, Atg7, but decreased the level of SQSTM1/P62 in response to cerebral IR injury, indicating that autophagic degradation was activated. As shown in Fig. 3, the LC3B-II expression increased significantly at 12 hours after reperfusion in IR group compared with Sham
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group. In addition, the expression level of LC3B-II in IR + RIPoC group further increased and was higher than that in IR group. This result was
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further confirmed by immunofluorescence analysis (Fig. 3). Similar results
were obtained for the expression of Beclin-1 and Atg7. We then examined
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the level of SQSTM1/P62, which is degraded in autophagolysosomes. By
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contrast, the SQSTM1/P62 protein expression levels were lower in the IR
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+ RIPoC group than those in the IR group.
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RIPoC protected against cerebral IR injury via induction of autophagy
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To clarify the involvement of activated autophagy in RIPoC-mediated
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neuroprotection, autophagy was blocked with the autophagy inhibitor 3methyladenine (3-MA). 3-MA injection before RIPoC treatment abolished its neuroprotective effects as evidenced by the reversed NDS, brain water
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content, and infarct volume (Fig. 4). The inhibitory effect of 3-MA on autophagy was confirmed by western blot detection of LC3B, Beclin-1, Atg7, and SQSTM1/P62. 3-MA treatment decreased LC3B-II, Beclin-1, and Atg7 expression and increased SQSTM1/P62 expression significantly, 15
indicating that autophagy process was suppressed (Fig. 4). The expression levels of Bax, Bcl-2, and active caspase-3 were also measured using western blot. Compared with MCAO + RIPoC group, 3-MA significantly increased expression levels of Bax and active caspase-3, and decreased
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Bcl-2 expression level. TUNEL staining indicated that 3-MA abolished the
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anti-apoptotic effect of RIPoC (Fig. 4).
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RIPoC induced AMPK pathway during cerebral IR injury
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To explore the involvement of AMPK pathway in RIPoC-mediated
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neuroprotection, we detected the expression levels of p-AMPK/AMPK, p-
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ACC/ACC, p-mTOR/mTOR, p-ULK1/ULK1 by Western blot. As shown
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in Fig. 5, RIPoC treatment significantly increased the ratios of pAMPK/AMPK, p-ACC/ACC, and p-ULK1/ULK1, while decreased the
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ratio of p-mTOR/mTOR, indicating that AMPK pathway was activated by
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RIPoC treatment.
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RIPoC-induced autophagy was dependent on AMPK pathway
To investigate the role of AMPK in RIPoC-induced autophagy process, CC was injected at the onset of reperfusion. After 12 h reperfusion, the pAMPK /AMPK, p-ULK1/ULK1, and p-ACC /ACC ratios in the cerebral 16
cortex of the right hemisphere were detected by Western blot. CC markedly decreased the ratios of p-AMPK /AMPK, p-ULK1/ULK1, and p-ACC /ACC, and increased the ratio of p-mTOR/mTOR (Fig. 6). More importantly, CC significantly attenuated the RIPoC-induced autophagy, as
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evidenced by decreased levels of LC3B-II/LC3B- I, Beclin-1, and Atg7, and increased level of p62. Moreover, immunofluorescence also showed
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that CC decreased the expression of LC3B (Fig. 6).
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Discussion
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The major findings we documented in the present study are: (1) RIPoC
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confers neuroprotection against cerebral IR injury by inducing autophagy
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via AMPK pathway. (2) RIPoC attenuates infarct size, neurological deficit and brain edema after cerebral IR injury. (3) The beneficial effects induced
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by RIPoC are depended on the activation of autophagy. (4) AMPK pathway
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is critical for the activation of autophagy in RIPoC-induced neuroprotection. In the current study, we observed the up-regulation of autophagy caused by RIPoC, as indicated by the increased expression of
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autophagy markers LC3B-II, Beclin-1, and Atg7 as well as the degradation of SQSTM1/P62. To clarify the contribution of autophagy to the RIPoCinduced neuroprotection, 3-MA, a widely used autophagy inhibitor [24], was administered prior to the IR injury and RIPoC treatment to block 17
macroautophagy component of lysosomal activity. Indeed, 3-MA administration dissected the autophagy flux and abolished the neuroprotection induced by RIPoC, indicating that autophagy is an essential element of protection induced by RIPoC against IR injury. To
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further determine the regulatory mechanisms of autophagy, we detected the phosphorylation level of AMPK, mTOR, ACC, and ULK1. AMPK, ACC,
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ULK1 were activated while mTOR was deactivated by RIPoC treatment. Then, CC, a common AMPK inhibitor, was used to block AMPK pathway.
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Notably, the inhibition of AMPK partially prevented RIPoC-induced
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autophagy and attenuated the protection effects of RIPoC. AMPK is
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supposed to promote autophagy not only by directly activating ULK1 but
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also by negatively regulating mTOR and its inhibitory effect on ULK1 [8].
autophagy
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The potential signaling mechanisms involved in AMPK-induced might
be
regulating
autophagy
through
coordinated
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phosphorylation of ULK1. Taken together, these findings support that
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RIPoC-induced autophagy is mediated, at least in part, by AMPK signaling pathway.
Clearly, timely and complete reperfusion is the most effective way to
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protect the brain against ischemia. However, reperfusion also causes an additional reperfusion injury on top of ischemic injury, and it, thus, contributes to infarction. Therefore, adjunct interventions and treatments on top of timely reperfusion are urgently needed to reduce infarct size and 18
improve the clinical outcome in patients with AIS. Ischemic conditioning strategies are considered promising therapies for cerebral ischemia [11]. The protection of IPreC has been shown in ischemic stroke patients, such as reported by a clinical study that transient ischemic attacks (TIA) before
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ischemic stroke could induce tolerance, alleviate neurological deficits, and improve outcomes of acute stroke patients [33]. However, compared with
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local ischemic conditioning, which is limited to cerebral angioplasty patients, remote ischemic conditioning (RIC) is much easier to perform in
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the setting of AIS. Therefore, remote ischemic conditioning was termed as
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the dawn in the darkness for acute ischemic stroke by some neuroscientists
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[23]. A clinical study has proved that RIPreC could improve cerebral
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perfusion and reduce recurrent strokes in patients with intra-arterial
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stenosis [22]. Moreover, prehospital RIPerC is also supposed to take immediate neuroprotective effects [12]. Nevertheless, the major
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disadvantage of ischemic preconditioning as a neuroprotective strategy is
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the requirement to intervene before the occurrence of the ischemic event, which is not possible in the case of AIS. Recently, RECAST (Remote Ischemic Conditioning After Stroke Trial) also confirmed RIC after AIS
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could improve neurological outcome and was safe and feasible [6]. The mechanisms by which these approaches provide neuroprotection are not fully elucidated, especially in autophagy. In terms of RIPoC, previous studies have demonstrated that RIPoC could reduce IR injury by 19
inhibiting oxidative stress via NADPH [3] and Nrf2-ARE pathway [17]. RIPoC is also proved to reduce neuronal apoptosis and inflammation through the activation of STAT3 [4]. Recently, there is growing interest in investigating the role of autophagy in these processes. The role of
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autophagy in cerebral ischemia is still controversial. Some studies show that activation of autophagy is detrimental [34, 36, 38], while others
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support a neuroprotective role [1, 21, 29]. Several ischemic conditioning
strategies have been demonstrated to activate autophagy in the ischemic
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heart, liver, and brain [10, 19, 26]. RIPoC is proved to be able to induce
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autophagy, which plays a key role in protecting against IR injury in other
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organ [10]. However, the knowledge of autophagy in RIPoC-afforded
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protection and its regulatory mechanisms in cerebral IR injury is still
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limited. Autophagy activation contributes to the neuroprotection by IPreC and RIPerC protects against focal cerebral ischemia in rats respectively [13, Recently, several reports have demonstrated that the protective
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30].
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effects of RIPoC are associated with autophagy induction and the reduction of mitochondrial damage after cerebral ischemia in rats [26, 27]. These findings indicate autophagy plays a crucial role in RIPoC-induced
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neuroprotection with the involvement of AKT/GSK3β and Bcl-2 activation [26]. In contrast, Wang et al. [32] demonstrate that RIPerC + IPoC presented neuroprotection via inhibiting the autophagy process. IPoC was considered to protect against cerebral ischemia by inhibiting autophagy in 20
rats in another study [7]. The difference of results among these studies and ours may be mainly due to the different models and different ways of blocking autophagy. Zhang et al. [37] indicate that autophagy plays different roles in different phases of cerebral ischemia. Interestingly,
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inhibition of autophagy protects against permanent ischemia in brain but aggravates cerebral IR injury. We hypothesize that autophagy could be a
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double-edged sword while IC might be a dual-directional regulation treatment on autophagy, and further studies are still needed to address this
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issue.
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This study also has some limitations. Although infarct size and cerebral
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function were preserved by RIPoC, the potential mechanisms and
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protective factors involved in transmitting the signal from the limbs to the
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brain were not fully explored in the study. Signal transmission to the brain may be dependent on interactions between humoral and neural pathways
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[11]. The levels of serum protective factors need to be comprehensively
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evaluated in further studies. In summary, we documented that RIPoC could ameliorate cerebral IR
injury, and the protective mechanism appeared to involve its ability to
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induce autophagy via AMPK pathway. These findings provide new insights into the linkage between RIPoC and autophagy signaling under ischemic conditions, suggesting that AMPK is a potential regulator of RIPoC-induced autophagy. Moreover, induction of autophagy by RIPoC 21
may be a promising adjunct therapeutic strategy in stroke patients.
Acknowledgements This study was supported by grants from the National Natural Science
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Foundation of China (81630027, 81571215,), Leading Talents of Tangdu Hospital, and Leading Talents of Middle Age and Young in S&T
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Innovation supported by Chinese Science and Technology Ministry
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(2013RA2181).
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Figure Legends:
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Fig. 1 Experimental protocol. IR ischemia-reperfusion, RIPoC remote limb
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ischemic postconditioning, Veh vehicle, 3-MA 3-methyladenine, CC compound C
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Fig. 2 RIPoC protected against cerebral IR injury. (A) Representative TTCstained brain slices were shown. White region presents the infarct area, while red region presents the normal brain area. (B) Infarct volumes were measured by TTC staining 12 h after reperfusion. (C) Neurological deficit 29
scores (NDS) were assessed in each group 12 h after reperfusion. (D) Brain water content were measured 12 h after reperfusion. (E) Representative western blot images of active caspase-3, Bax, and Bcl-2. (F) Active caspase-3 levels. (G) Bax levels. (H) Bcl-2 levels. (I) Typical images of
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TUNEL staining. Scale bar = 20 μm. Data are presented as mean ± standard deviation (SD), n=6 per group. *P < 0.05, versus the sham group. # P <
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0.05, versus the IR group. IR ischemia-reperfusion, RIPoC remote limb
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ischemic postconditioning
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Fig. 3 RIPoC induced autophagy during cerebral IR injury. (A)
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Representative western blot images of autophagy markers LC3B, Beclin-
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1, Atg 7, and SQSTM1/P62. (B) LC3BII levels. (C) Beclin-1 levels. (D)
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Atg 7 levels. (E) SQSTM1/P62 levels. (F) Typical images of immunofluorescence staining of LC3-II were shown (×800). Brain sections
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were double-labeled by DAPI (blue) and LC3B (green). Data are presented
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as mean ± SD, n=6 in each group. * P < 0.05, versus the sham group. # P < 0.05, versus the IR group.
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Fig. 4 RIPoC protected against cerebral IR injury via induction of autophagy. (A) Representative TTC-stained brain slices were shown. White region presents the infarct area, while red region presents the normal brain area. (B) Infarct volumes were measured by TTC staining 12 h after 30
reperfusion. (C) Neurological deficit scores (NDS) were assessed in each group 12 h after reperfusion. (D) Brain water content were measured 12 h after reperfusion. (E) Representative western blot images of active caspase3, Bax, and Bcl-2. (F) Active caspase-3 levels. (G) Bax levels. (H) Bcl-2
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levels. (I) Representative western blot images of autophagy markers LC3B, Beclin-1, Atg 7, and SQSTM1/P62. (J) LC3BII levels. (K) Beclin-1 levels.
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(L) Atg 7 levels. (M) SQSTM1/P62 levels. (N) Typical images of TUNEL staining. Scale bar = 20μm. Data are presented as mean ± SD, n=6 in each
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group. & P < 0.05, versus the IR + Veh group. # P < 0.05, versus the IR+
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Veh +RIPoC group. Veh vehicle, 3-MA 3-methyladenine
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Fig. 5 RIPoC induced AMPK pathway during cerebral IR injury. (A)
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Representative western blot images of p-AMPK, AMPK, ULK1, p-ULK1, ACC, p-ACC, mTOR, p-mTOR. (B) Ratio of p-AMPK/AMPK. (C) Ratio
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of p-ULK1/ULK1. (D) Ratio of p-ACC/ACC. (E) Ratio of p-
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mTOR/mTOR. Data are presented as mean ± SD, n=6 in each group. * P < 0.05, versus the sham group. # P < 0.05, versus the IR group.
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Fig. 6 RIPoC-induced autophagy was dependent on AMPK pathway. (A) Representative western blot images of autophagy markers LC3B, Beclin1, Atg 7, and SQSTM1/P62. (B) LC3BII levels. (C) Beclin-1 levels. (D) Atg 7 levels. (E) SQSTM1/P62 levels. (F) Typical images of 31
immunofluorescence staining of LC3-II were shown (×800). Brain sections were double-labeled by DAPI (blue) and LC3B (green). (G) Representative western blot images of p-AMPK, AMPK, ULK1, p-ULK1, ACC, p-ACC, mTOR, p-mTOR. (H) Ratio of p-AMPK/AMPK. (I) Ratio of p-
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ULK1/ULK1. (J) Ratio of p-ACC/ACC. (K) Ratio of p-mTOR/mTOR. Data are presented as mean ± SD, n=6 per group. & P < 0.05, versus the IR
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+ Veh group. # P < 0.05, versus the IR+ Veh +RIPoC group. Veh vehicle,
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S0361-9230(17)30605-6 https://doi.org/10.1016/j.brainresbull.2018.02.013 BRB 9380
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
16-10-2017 26-1-2018 9-2-2018
Please cite this article as: Hao Guo, Lei Zhao, Bodong Wang, Xia Li, Hao Bai, Haixiao Liu, Liang Yue, Wei Guo, Zhenyuan Bian, Li Gao, Dayun Feng, Yan Qu, Remote limb ischemic postconditioning protects against cerebral ischemiareperfusion injury by activating AMPK-dependent autophagy, Brain Research Bulletin https://doi.org/10.1016/j.brainresbull.2018.02.013 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.
Remote limb ischemic postconditioning protects against cerebral ischemia-reperfusion injury by activating AMPKdependent autophagy
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Hao Guo a, 1, Lei Zhao a, 1, Bodong Wang a, b, 1, Xia Li a, Hao Bai a, Haixiao Liu a, Liang Yue a, c, Wei Guo a, Zhenyuan Bian d, Li Gao a, Dayun Feng a,
a
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Yan Qu a, *
Department of Neurosurgery, Tangdu Hospital, The Fourth Military
Department of Neurosurgery, Jinan Military General Hospital, Jinan
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b
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Medical University, Xi’an 710038, China
Department of Neurosurgery, Xi’an Aerospace General Hospital, Xi’an
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c
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250031, China
d
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710100, China
Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth
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Military Medical University, Xi’an 710032, China
1
These authors contribute equally to this work.
*
Corresponding author: Yan Qu, Department of Neurosurgery, Tangdu
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Hospital, Fourth Military Medical University, No. 569 Xinsi Road, Xi’an 710038, China. E-mail: [email protected].
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Highlights: RIPoC markedly decreases neuronal injury and infarct size after cerebral I/R.
RIPoC alleviates neuronal apoptosis after cerebral I/R.
RIPoC activates AMPK-dependent autophagy after cerebral I/R.
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Abstract
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Remote limb ischemic postconditioning (RIPoC) is a promising adjunct treatment for cerebral ischemia-reperfusion (IR) injury. However, the
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underlying mechanisms have not been fully elucidated yet. The present
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study aims to investigate potential involvement and regulatory mechanisms
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of autophagy in RIPoC treatment against cerebral IR injury in mice. Mice
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were subjected to 2 h middle cerebral artery occlusion (MCAO) then
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treated with vehicle, 3-methyladenine (3-MA, an autophagy inhibitor), or compound C (an AMPK inhibitor) at the onset of reperfusion. RIPoC was
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carried out by 3 cycles of 10-min occlusion-reperfusion of bilateral femoral
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artery at the beginning of the reperfusion. Infarct volume, neurological score, and brain water content of the mice were assessed after 12 h reperfusion. Autophagy markers, cell apoptosis markers, and AMPK
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pathway activity were also evaluated. Our results indicated that RIPoC treatment reduced neurological deficits, brain water content, and infarct volume after IR. Meanwhile, RIPoC was proved to induce autophagy and activate AMPK pathway. Furthermore, the RIPoC-induced autophagy and 2
neuroprotection were abolished by 3-MA and partially blocked by compound C. In conclusion, the present study suggests that RIPoC attenuates cerebral IR injury by activating AMPK-dependent autophagy.
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Key words: remote limb ischemic postconditioning, cerebral ischemia-
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reperfusion injury, AMPK, autophagy
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Introduction
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Stroke is one of the most common diseases with a leading cause of high
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morbidity and mortality worldwide [28]. Over 80% of all strokes are
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ischemic which were caused by insufficient blood supply to the brain.
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Novel therapeutic methods and medical devices have decreased the mortality and disability rate of acute ischemic stroke (AIS) over the last
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decades [2]. Although Solitaire stent retriever and alteplase (rtPA) have
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been approved by the FDA for the treatment of AIS, the outcome remains unsatisfactory due to the narrow time window, side effects, and some other factors [14]. Besides, despite the success of reperfusion therapy by
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endovascular treatment and thrombolysis, the ischemia-reperfusion (IR) injury is still of significant concern [9]. Therefore, adjunct therapies on top of timely and effective reperfusion are still needed to improve the clinical outcome and reduce the infarct size of patients with AIS. 3
Ischemic conditioning (IC) encompasses a number of related endogenous neuroprotective strategies which conduct a series of transient periods of mechanical occlusions and reperfusions to protect against vital organ ischemia [15]. It can be applied either directly to the target organ or
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from afar, for example limbs. Remote limb ischemic postconditioning (RIPoC) is an efficacious protective strategy which performs ischemic
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condition on a remote organ (limbs) after the ischemic period of the brain
to protect the brain against IR injury in experimental stroke. It has attracted
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extensive interest recently. Although the neuroprotection of RIPoC has
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been successfully proved in several studies, the underlying mechanisms
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have not been fully elucidated yet [3, 25].
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Autophagy is an evolutionarily conserved process in which
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unnecessary or dysfunctional proteins and organelles are delivered to the lysosomes for degradation [24]. As a vital degradation/recirculation system
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in at the cellular level, autophagy is considered to play an important role in
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many pathological conditions, including AIS [37]. Although it is undisputable that autophagy can be activated after cerebral ischemia, whether autophagy plays a protective or detrimental role in IR injury
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remains controversial [31]. AMP-activated protein kinase (AMPK) is a highly conserved crucial energy sensor which regulates cellular metabolism to maintain energy homeostasis [8]. AMPK signaling pathway also regulates autophagy as previously demonstrated [16]. In particularly, 4
AMPK has been proved to phosphorylate and activate unc-51-like autophagy-activating kinase 1 (ULK1), which triggers the initiation of the autophagic cascade [5]. In this investigation, we aim to explore the potential involvement and function of autophagy in RIPoC treatment
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of AMPK-dependent autophagy in RIPoC-induced protection.
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against cerebral IR injury in mice. Moreover, we also investigate the role
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Materials and methods
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Reagents
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Dimethyl sulfoxide (DMSO), 2,3,5-triphenyltetrazolium chloride
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(TTC), and 4’,6-diamino-2-phenylindole (DAPI) were purchased from
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Sigma-Aldrich (St. Louis, MO, USA). Bicinchoninic acid (BCA) protein assay kits were purchased from Beyotime Biotech Co. (Shanghai, China).
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3-Methyladenine (3-MA) was purchased from Selleckchem (Houston, TX,
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USA). Compound C (CC) (ab146597) and Rabbit polyclonal antibody against active caspase-3 (ab2302) was obtained from Abcam (Cambridge, UK). Rabbit monoclonal antibodies against Bcl-2 (#2870)，Bax (#14796) ,
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β-actin (#4970), LC3B (#3868), Beclin-1 (#3495), Atg7 (#8558), SQSTM1/P62 (#8025), AMPKα (#5831), Phosphor-AMPKα (Thr172) (#50081), mTOR (#2983), Phosphor-mTOR (Ser2448) (#5536), Acetyl-CoA Carboxylase (ACC) (#3676), Phosphor-ACC (Ser79) (#11818), unc-51-like 5
autophagy-activating kinase 1 (ULK1) (#8054), and Phosphor-ULK1 (Ser555) (#5869) were purchased from Cell Signaling Technology (Beverly, MA, USA). The HRP-conjugated rabbit anti-mouse secondary antibody and HRP-conjugated goat anti-rabbit secondary antibody were purchased
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from Bioworld Co. (Shanghai, China). Terminal deoxynucleotidyl transferased UTP nick-end labeling (TUNEL) kit was purchased from
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Roche (Mannheim, Germany).
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Experimental animals
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All animals used in this study received humane care according to the
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Guide for the Care and Use of Laboratory Animals, which was published
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by the US National Institutes of Health (National Institutes of Health
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Publication no. 85-23, revised 1996). All protocols were approved by the Ethics Committee of the Fourth Military Medical University.
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Healthy adult male C57BL/6 J mice weighing 20-25g were purchased
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from the Experimental Animal Center of the Fourth Military Medical University. The mice were kept in a pathogen-free environment at a constant temperature (23 °C) and humidity (60 %), with a 12-h light/dark
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cycle and had free access to food and water for two weeks before the experiment as an acclimatization period.
Induction of MCAO 6
Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) as described previously [35]. Briefly, mice were anesthetized with a mixture of isoflurane (3 % induction, 1.5 % maintenance, v/v) in 30 % oxygen and 70 % nitrous oxide. Heating lamps
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were used to maintain temperature at 36.5-37 °C. A midline neck incision was prepared. Then, the right common carotid artery (CCA), internal
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carotid artery (ICA) and external carotid artery (ECA) were exposed and isolated under an operating microscope. After the CCA was ligated close
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to its origin with a 3-0 silk suture, a 6-0 nylon monofilament suture (Beijing
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Sunbio Biotech Co. Ltd, Beijing, China), blunted and coated with a
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rounded tip, was inserted through a small incision into the CCA and
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advanced 10 mm distal to the carotid bifurcation until mild resistance to
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occlude the origin of the MCA. Reperfusion was allowed after 2 h of MCAO by monofilament removal. Sham-operated animals received
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not cut.
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midline neck incisions. The right common carotid artery was isolated, but
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Induction of RIPoC RIPoC was induced using an open invasive technique after 2 h of
MCAO. Under anesthesia, right femoral artery was exposed. RIPoC was induced by three cycles of 10-min of bilateral femoral arteries occlusion by a microvascular clamp to occlude the femoral vessels under an operating 7
microscope, followed by 10-min of reperfusion. Bilateral distal limb pallor were observed during occlusion, followed rapidly by brisk reactive hyperemia during reperfusion. Anesthesia was discontinued and animals were placed back into their cages. In the sham and IR model groups, the
Drug administration Intracerebroventricular
(ICV)
injection
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femoral arteries were only exposed and isolated.
was
performed
under
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isoflurane anesthesia. Mice were placed on a stereotaxic apparatus with the
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skull held horizontally. 3-MA and CC were dissolved in DMSO separately.
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Mice were administered an ICV injection of vehicle or 3-MA or CC at the
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onset of reperfusion. Control mice were injected with the same volume of
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DMSO. Vehicle, 3-MA, or CC was infused 0.5 μL/min into lateral ventricle using a 10 µL Hamilton syringe at the following coordinate: 0.46 mm
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rostral, 1mm lateral in relation to bregma, and 2.8 mm depth from the
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surface. The needle was kept in place for 10 min after injection completion and then withdrawn slowly.
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Experimental design Mice were randomized divided into the following groups (Fig. 1). 1. Sham group, in which mice were subjected to the same as surgical operation, but without occlusion of the right middle cerebral artery and 8
RIPoC treatment. 2. IR group, in which mice were subjected to 2 h of MCAO followed by reperfusion. 3. IR + RIPoC group, in which mice received bilateral hindlimb
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intervention with 3 cycles of 10-min occlusion-reperfusion immediately at the onset of reperfusion.
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4. IR + Veh group, in which mice were administered vehicle by ICV injection at the onset of reperfusion.
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5. IR + Veh + RIPoC group, in which mice were administered vehicle by
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ICV injection and bilateral hindlimb intervention with 3 cycles of 10-min
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occlusion-reperfusion at the onset of reperfusion.
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6. IR + 3-MA group, in which mice were administered 3-MA by ICV
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injection at the onset of reperfusion.
7. IR + 3-MA + RIPoC group, in which mice were administered 3-MA by
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ICV injection and bilateral hindlimb intervention with 3 cycles of 10-min
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occlusion-reperfusion at the onset of reperfusion. 8. IR + CC group，in which mice were administered CC by ICV at the onset of reperfusion.
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9. IR+ CC + RIPoC group, in which mice were administered CC by ICV injection and bilateral hindlimb intervention with 3 cycles of 10-min occlusion-reperfusion at the onset of reperfusion.
9
Measurement of neurological deficit and cerebral edema The mice were returned to their cages after the procedures were finished and again allowed free access to food and water. The neurological deficit score (NDS) was assessed just before sacrifice at 12 h after
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reperfusion as described previously [35]. Each mouse was scored by three examiners who were blinded to the identity of the mice or treatment
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protocol. NDS were evaluated as follows: 0, no deficit; 1, forelimb weakness and torso turning to the ipsilateral side when held by tail; 2,
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circling to the contralateral side but normal posture at rest; 3, unable to bear
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weight on the affected side at rest and 4, no spontaneous motor activity or
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barrel rolling. If no deficit was observed 2 h after the beginning of the
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occlusion period, the animal was removed from further study.
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The water content of brain was assessed at 12 h after reperfusion. As described previously [35], mice were decapitated under deep anesthesia.
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The brains were quickly removed and the hemispheres were separated in
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the sagittal plane. After the wet weight of the brain tissues was quantified, the tissues were desiccated at 105°C for 48 h until the weight was constant (dry weight). The water content of each brain was calculated as follow:
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(wet weight - dried weight) /wet weight × 100 %.
Measurement of infarct volume After a reperfusion period of 12 h, the cerebral infarct area was 10
identified by TTC staining as described previously [35]. The mice were sacrificed by rapid decapitation under deep anesthesia. The whole brain was rapidly removed and cut into 2-mm-thick coronal sections, then the slices were stained with 2 % solution of TTC diluted in 0.1 M sodium
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phosphate buffered saline (PBS) (pH 7.4) at 37 °C for 30-min in the dark and immersed 30-min in 10 % formalin. The infarct volume was calculated
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according to the following formula: V = t × (A1 + A2... + An), in which ‘t’ is the brain slice thickness and ‘A’ is the infarct area. The percentage
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cerebral infarct volume was measured as follows: (cerebral infarct total
A
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volume/whole brain volume) ×100 %.
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Western blot
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Western blot was performed as described previously [20]. After a reperfusion period of 12 h, mice were sacrificed under deep anesthesia and
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perfused with saline solution. The cerebral cortex from the right
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hemisphere of each mouse was homogenized and the total proteins were extracted by RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentrations were determined by BCA protein assay kit. Equal amounts
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of protein (40μg) were separated by 10-15% sodium dodecyl sulfatepolyacrylamide gelelectrophoresis (SDS-PAGE) gels electrophoresis and then transferred to PVDF membranes. The membranes were blocked by 2% bull serum albumin (BSA) in Tris-buffered saline and Tween 20 (pH 7.6) 11
(TBST) for 1 h at room temperature and were then incubated overnight at 4 °C with antibodies against Bax, Bcl-2, active caspase-3, LC3B, Beclin1, Atg7, SQSTM1/P62, AMPK, p-AMPK, mTOR, p-mTOR, ACC, p-ACC, ULK 1, p-ULK 1, and β-actin at 4°C overnight. The membranes were then
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incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:20,000 dilution) at room temperature for 2 h and washed with
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TBST. The protein bands were detected using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA) and quantified using the Quantity One
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software package (West Berkeley, CA, USA).
apoptosis
in the tissue was detected
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Cell
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TUNEL staining
using terminal
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deoxynucleotidyl transferased UTP nick end labeling (TUNEL) staining 12 h after cerebral IR induction in accordance with the manufacturer's
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instructions [18]. The samples were treated with 0.3% hydrogen peroxide
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for 30 minutes and incubated with 0.25% proteinase K for 45 minutes at 37ᵒC. Then, the slices were dyed using TUNEL reaction solution prepared according to the manufacturer instructions in a humidified dark chamber
A
for 1 h at 37ᵒC. Finally, the tissues were dyed using DAPI for 10 minutes at 37ᵒC. Meanwhile, each of these steps was followed by three 5-minute PBS washes. Sections were covered using cover glasses with anti-fade mounting medium. The TUNEL-positive cells showed green fluorescence 12
and nucleus were stained with blue fluorescence. Apoptotic index was determined as the ratio of the number of TUNEL-positive neurons to the total number of neurons. Five visual fields were randomly chosen from the ischemic penumbra of each slice under a high-power lens for counting
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apoptotic cells.
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Immunofluorescence
The sections were stained with rabbit monoclonal antibody against
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LC3B (1:200) at 4 ℃ , followed by goat anti-rabbit FITC-conjugated
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conjugated secondary antibody (CW0114, ComWin Biotech Co., Beijing,
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China) for 2 hours at room temperature. Then, the sections were incubated
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with DAPI staining solution for 10 min at room temperature. Photographs
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were snapped with a confocal fluorescent microscope (Nikon Eclipse Ti).
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Statistical analysis
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GraphPad Prism 6 was used to analyze data in this study. Data were presented as the mean ± SD. NDSs were analyzed by the non-parametric Kruskal-Wallis test. Other data were tested using one-way ANOVA. Tukey
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post hoc analysis was used for intergroup comparisons. P<0.05 was considered statistically significant.
Results 13
RIPoC protected against cerebral IR injury
To determine whether RIPoC could attenuate IR injury in ischemic brain,
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mice were subjected to 10 minutes of ischemia followed by 10 minutes of reperfusion on bilateral hindlimb immediately at the onset of cerebral
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reperfusion. NDS, infarct volumes, and cerebral edema were assessed 12 h after the IR insult. Based on these measures, NDS, brain water content, and
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infarct volumes increased dramatically after MCAO. RIPoC treatment
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significantly reduced NDS, brain water content, and infarct volume.
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Similar results were obtained for TUNEL staining (Fig. 2). The levels of
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Bax, active caspase-3, and Bcl-2 were measured using western blot. As
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shown in Fig. 2, the levels of Bax and active caspase-3 were significantly lower in the IR + RIPoC group than those levels in the IR group. By
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contrast, the RIPoC dramatically increased Bcl-2 expression level in the IR
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+ RIPoC group compared with the IR group (Fig. 2).
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RIPoC induced autophagy during cerebral IR injury
To determine the effect of RIPoC on autophagy in cerebral IR process, the levels of the autophagy markers LC3B, Beclin-1, Atg7, and SQSTM1/ P62 were detected. We found that RIPoC significantly increased the levels of 14
LC3B-II/LC3B-I, Beclin-1, Atg7, but decreased the level of SQSTM1/P62 in response to cerebral IR injury, indicating that autophagic degradation was activated. As shown in Fig. 3, the LC3B-II expression increased significantly at 12 hours after reperfusion in IR group compared with Sham
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group. In addition, the expression level of LC3B-II in IR + RIPoC group further increased and was higher than that in IR group. This result was
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further confirmed by immunofluorescence analysis (Fig. 3). Similar results
were obtained for the expression of Beclin-1 and Atg7. We then examined
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the level of SQSTM1/P62, which is degraded in autophagolysosomes. By
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contrast, the SQSTM1/P62 protein expression levels were lower in the IR
M
A
+ RIPoC group than those in the IR group.
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RIPoC protected against cerebral IR injury via induction of autophagy
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To clarify the involvement of activated autophagy in RIPoC-mediated
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neuroprotection, autophagy was blocked with the autophagy inhibitor 3methyladenine (3-MA). 3-MA injection before RIPoC treatment abolished its neuroprotective effects as evidenced by the reversed NDS, brain water
A
content, and infarct volume (Fig. 4). The inhibitory effect of 3-MA on autophagy was confirmed by western blot detection of LC3B, Beclin-1, Atg7, and SQSTM1/P62. 3-MA treatment decreased LC3B-II, Beclin-1, and Atg7 expression and increased SQSTM1/P62 expression significantly, 15
indicating that autophagy process was suppressed (Fig. 4). The expression levels of Bax, Bcl-2, and active caspase-3 were also measured using western blot. Compared with MCAO + RIPoC group, 3-MA significantly increased expression levels of Bax and active caspase-3, and decreased
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Bcl-2 expression level. TUNEL staining indicated that 3-MA abolished the
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anti-apoptotic effect of RIPoC (Fig. 4).
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RIPoC induced AMPK pathway during cerebral IR injury
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To explore the involvement of AMPK pathway in RIPoC-mediated
A
neuroprotection, we detected the expression levels of p-AMPK/AMPK, p-
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ACC/ACC, p-mTOR/mTOR, p-ULK1/ULK1 by Western blot. As shown
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in Fig. 5, RIPoC treatment significantly increased the ratios of pAMPK/AMPK, p-ACC/ACC, and p-ULK1/ULK1, while decreased the
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ratio of p-mTOR/mTOR, indicating that AMPK pathway was activated by
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RIPoC treatment.
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RIPoC-induced autophagy was dependent on AMPK pathway
To investigate the role of AMPK in RIPoC-induced autophagy process, CC was injected at the onset of reperfusion. After 12 h reperfusion, the pAMPK /AMPK, p-ULK1/ULK1, and p-ACC /ACC ratios in the cerebral 16
cortex of the right hemisphere were detected by Western blot. CC markedly decreased the ratios of p-AMPK /AMPK, p-ULK1/ULK1, and p-ACC /ACC, and increased the ratio of p-mTOR/mTOR (Fig. 6). More importantly, CC significantly attenuated the RIPoC-induced autophagy, as
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evidenced by decreased levels of LC3B-II/LC3B- I, Beclin-1, and Atg7, and increased level of p62. Moreover, immunofluorescence also showed
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that CC decreased the expression of LC3B (Fig. 6).
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Discussion
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The major findings we documented in the present study are: (1) RIPoC
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confers neuroprotection against cerebral IR injury by inducing autophagy
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via AMPK pathway. (2) RIPoC attenuates infarct size, neurological deficit and brain edema after cerebral IR injury. (3) The beneficial effects induced
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by RIPoC are depended on the activation of autophagy. (4) AMPK pathway
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is critical for the activation of autophagy in RIPoC-induced neuroprotection. In the current study, we observed the up-regulation of autophagy caused by RIPoC, as indicated by the increased expression of
A
autophagy markers LC3B-II, Beclin-1, and Atg7 as well as the degradation of SQSTM1/P62. To clarify the contribution of autophagy to the RIPoCinduced neuroprotection, 3-MA, a widely used autophagy inhibitor [24], was administered prior to the IR injury and RIPoC treatment to block 17
macroautophagy component of lysosomal activity. Indeed, 3-MA administration dissected the autophagy flux and abolished the neuroprotection induced by RIPoC, indicating that autophagy is an essential element of protection induced by RIPoC against IR injury. To
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further determine the regulatory mechanisms of autophagy, we detected the phosphorylation level of AMPK, mTOR, ACC, and ULK1. AMPK, ACC,
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ULK1 were activated while mTOR was deactivated by RIPoC treatment. Then, CC, a common AMPK inhibitor, was used to block AMPK pathway.
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Notably, the inhibition of AMPK partially prevented RIPoC-induced
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autophagy and attenuated the protection effects of RIPoC. AMPK is
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supposed to promote autophagy not only by directly activating ULK1 but
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also by negatively regulating mTOR and its inhibitory effect on ULK1 [8].
autophagy
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The potential signaling mechanisms involved in AMPK-induced might
be
regulating
autophagy
through
coordinated
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phosphorylation of ULK1. Taken together, these findings support that
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RIPoC-induced autophagy is mediated, at least in part, by AMPK signaling pathway.
Clearly, timely and complete reperfusion is the most effective way to
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protect the brain against ischemia. However, reperfusion also causes an additional reperfusion injury on top of ischemic injury, and it, thus, contributes to infarction. Therefore, adjunct interventions and treatments on top of timely reperfusion are urgently needed to reduce infarct size and 18
improve the clinical outcome in patients with AIS. Ischemic conditioning strategies are considered promising therapies for cerebral ischemia [11]. The protection of IPreC has been shown in ischemic stroke patients, such as reported by a clinical study that transient ischemic attacks (TIA) before
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ischemic stroke could induce tolerance, alleviate neurological deficits, and improve outcomes of acute stroke patients [33]. However, compared with
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local ischemic conditioning, which is limited to cerebral angioplasty patients, remote ischemic conditioning (RIC) is much easier to perform in
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the setting of AIS. Therefore, remote ischemic conditioning was termed as
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the dawn in the darkness for acute ischemic stroke by some neuroscientists
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[23]. A clinical study has proved that RIPreC could improve cerebral
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perfusion and reduce recurrent strokes in patients with intra-arterial
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stenosis [22]. Moreover, prehospital RIPerC is also supposed to take immediate neuroprotective effects [12]. Nevertheless, the major
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disadvantage of ischemic preconditioning as a neuroprotective strategy is
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the requirement to intervene before the occurrence of the ischemic event, which is not possible in the case of AIS. Recently, RECAST (Remote Ischemic Conditioning After Stroke Trial) also confirmed RIC after AIS
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could improve neurological outcome and was safe and feasible [6]. The mechanisms by which these approaches provide neuroprotection are not fully elucidated, especially in autophagy. In terms of RIPoC, previous studies have demonstrated that RIPoC could reduce IR injury by 19
inhibiting oxidative stress via NADPH [3] and Nrf2-ARE pathway [17]. RIPoC is also proved to reduce neuronal apoptosis and inflammation through the activation of STAT3 [4]. Recently, there is growing interest in investigating the role of autophagy in these processes. The role of
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autophagy in cerebral ischemia is still controversial. Some studies show that activation of autophagy is detrimental [34, 36, 38], while others
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support a neuroprotective role [1, 21, 29]. Several ischemic conditioning
strategies have been demonstrated to activate autophagy in the ischemic
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heart, liver, and brain [10, 19, 26]. RIPoC is proved to be able to induce
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autophagy, which plays a key role in protecting against IR injury in other
A
organ [10]. However, the knowledge of autophagy in RIPoC-afforded
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protection and its regulatory mechanisms in cerebral IR injury is still
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limited. Autophagy activation contributes to the neuroprotection by IPreC and RIPerC protects against focal cerebral ischemia in rats respectively [13, Recently, several reports have demonstrated that the protective
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30].
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effects of RIPoC are associated with autophagy induction and the reduction of mitochondrial damage after cerebral ischemia in rats [26, 27]. These findings indicate autophagy plays a crucial role in RIPoC-induced
A
neuroprotection with the involvement of AKT/GSK3β and Bcl-2 activation [26]. In contrast, Wang et al. [32] demonstrate that RIPerC + IPoC presented neuroprotection via inhibiting the autophagy process. IPoC was considered to protect against cerebral ischemia by inhibiting autophagy in 20
rats in another study [7]. The difference of results among these studies and ours may be mainly due to the different models and different ways of blocking autophagy. Zhang et al. [37] indicate that autophagy plays different roles in different phases of cerebral ischemia. Interestingly,
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inhibition of autophagy protects against permanent ischemia in brain but aggravates cerebral IR injury. We hypothesize that autophagy could be a
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double-edged sword while IC might be a dual-directional regulation treatment on autophagy, and further studies are still needed to address this
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issue.
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This study also has some limitations. Although infarct size and cerebral
A
function were preserved by RIPoC, the potential mechanisms and
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protective factors involved in transmitting the signal from the limbs to the
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brain were not fully explored in the study. Signal transmission to the brain may be dependent on interactions between humoral and neural pathways
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[11]. The levels of serum protective factors need to be comprehensively
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evaluated in further studies. In summary, we documented that RIPoC could ameliorate cerebral IR
injury, and the protective mechanism appeared to involve its ability to
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induce autophagy via AMPK pathway. These findings provide new insights into the linkage between RIPoC and autophagy signaling under ischemic conditions, suggesting that AMPK is a potential regulator of RIPoC-induced autophagy. Moreover, induction of autophagy by RIPoC 21
may be a promising adjunct therapeutic strategy in stroke patients.
Acknowledgements This study was supported by grants from the National Natural Science
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Foundation of China (81630027, 81571215,), Leading Talents of Tangdu Hospital, and Leading Talents of Middle Age and Young in S&T
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Innovation supported by Chinese Science and Technology Ministry
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(2013RA2181).
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Figure Legends:
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Fig. 1 Experimental protocol. IR ischemia-reperfusion, RIPoC remote limb
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ischemic postconditioning, Veh vehicle, 3-MA 3-methyladenine, CC compound C
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Fig. 2 RIPoC protected against cerebral IR injury. (A) Representative TTCstained brain slices were shown. White region presents the infarct area, while red region presents the normal brain area. (B) Infarct volumes were measured by TTC staining 12 h after reperfusion. (C) Neurological deficit 29
scores (NDS) were assessed in each group 12 h after reperfusion. (D) Brain water content were measured 12 h after reperfusion. (E) Representative western blot images of active caspase-3, Bax, and Bcl-2. (F) Active caspase-3 levels. (G) Bax levels. (H) Bcl-2 levels. (I) Typical images of
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TUNEL staining. Scale bar = 20 μm. Data are presented as mean ± standard deviation (SD), n=6 per group. *P < 0.05, versus the sham group. # P <
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0.05, versus the IR group. IR ischemia-reperfusion, RIPoC remote limb
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ischemic postconditioning
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Fig. 3 RIPoC induced autophagy during cerebral IR injury. (A)
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Representative western blot images of autophagy markers LC3B, Beclin-
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1, Atg 7, and SQSTM1/P62. (B) LC3BII levels. (C) Beclin-1 levels. (D)
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Atg 7 levels. (E) SQSTM1/P62 levels. (F) Typical images of immunofluorescence staining of LC3-II were shown (×800). Brain sections
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were double-labeled by DAPI (blue) and LC3B (green). Data are presented
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as mean ± SD, n=6 in each group. * P < 0.05, versus the sham group. # P < 0.05, versus the IR group.
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Fig. 4 RIPoC protected against cerebral IR injury via induction of autophagy. (A) Representative TTC-stained brain slices were shown. White region presents the infarct area, while red region presents the normal brain area. (B) Infarct volumes were measured by TTC staining 12 h after 30
reperfusion. (C) Neurological deficit scores (NDS) were assessed in each group 12 h after reperfusion. (D) Brain water content were measured 12 h after reperfusion. (E) Representative western blot images of active caspase3, Bax, and Bcl-2. (F) Active caspase-3 levels. (G) Bax levels. (H) Bcl-2
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levels. (I) Representative western blot images of autophagy markers LC3B, Beclin-1, Atg 7, and SQSTM1/P62. (J) LC3BII levels. (K) Beclin-1 levels.
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(L) Atg 7 levels. (M) SQSTM1/P62 levels. (N) Typical images of TUNEL staining. Scale bar = 20μm. Data are presented as mean ± SD, n=6 in each
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group. & P < 0.05, versus the IR + Veh group. # P < 0.05, versus the IR+
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Veh +RIPoC group. Veh vehicle, 3-MA 3-methyladenine
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Fig. 5 RIPoC induced AMPK pathway during cerebral IR injury. (A)
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Representative western blot images of p-AMPK, AMPK, ULK1, p-ULK1, ACC, p-ACC, mTOR, p-mTOR. (B) Ratio of p-AMPK/AMPK. (C) Ratio
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of p-ULK1/ULK1. (D) Ratio of p-ACC/ACC. (E) Ratio of p-
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mTOR/mTOR. Data are presented as mean ± SD, n=6 in each group. * P < 0.05, versus the sham group. # P < 0.05, versus the IR group.
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Fig. 6 RIPoC-induced autophagy was dependent on AMPK pathway. (A) Representative western blot images of autophagy markers LC3B, Beclin1, Atg 7, and SQSTM1/P62. (B) LC3BII levels. (C) Beclin-1 levels. (D) Atg 7 levels. (E) SQSTM1/P62 levels. (F) Typical images of 31
immunofluorescence staining of LC3-II were shown (×800). Brain sections were double-labeled by DAPI (blue) and LC3B (green). (G) Representative western blot images of p-AMPK, AMPK, ULK1, p-ULK1, ACC, p-ACC, mTOR, p-mTOR. (H) Ratio of p-AMPK/AMPK. (I) Ratio of p-
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ULK1/ULK1. (J) Ratio of p-ACC/ACC. (K) Ratio of p-mTOR/mTOR. Data are presented as mean ± SD, n=6 per group. & P < 0.05, versus the IR
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+ Veh group. # P < 0.05, versus the IR+ Veh +RIPoC group. Veh vehicle,
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