Breviscapine confers a neuroprotective efficacy against transient focal cerebral ischemia by attenuating neuronal and astrocytic autophagy in the penumbra

Biomedicine & Pharmacotherapy 90 (2017) 69–76

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Breviscapine confers a neuroprotective efficacy against transient focal cerebral ischemia by attenuating neuronal and astrocytic autophagy in the penumbra Zhang Pengyue, Guo Tao, He Hongyun, Yang Liqiang, Deng Yihao* Department of morphology, Medical School, Kunming University of Science and Technology, Kunming 650500, China

A R T I C L E I N F O

Article history: Received 16 December 2016 Received in revised form 6 March 2017 Accepted 14 March 2017 Keywords: Breviscapine Cerebral ischemia Penumbra Autophagy inhibition Neuroprotection

A B S T R A C T

Breviscapine is a flavonoid derived from a traditional Chinese herb Erigerin breviscapus (Vant.) Hand-Mazz, and has been extensively used in clinical treatment for cerebral stroke in China, but the underlying pharmacological mechanisms are still unclear. In present study, we investigated whether breviscapine could confer a neuroprotection against cerebral ischemia injury by targeting autophagy mechanisms. A cerebral stroke model in Sprague-Dawley rats was prepared by middle cerebral artery occlusion (MCAO), rats were then randomly divided into 5 groups: MCAO + Bre group, rats were treated with breviscapine; MCAO + Tat-Beclin-1 group, animals were administrated with specific autophagy inducer Tat-Beclin-1; MCAO + Bre + Tat-Beclin-1 group, rats were treated with both breviscapine and Tat-Beclin-1, MCAO + saline group, rats received the same volume of physiological saline, and Sham surgery group. The autophagy levels in infarct penumbra were evaluated by western blotting, real-time PCR and immunofluorescence 7 days after the insult. Meanwhile, infarct volume, brain water content and neurological deficit score were assessed. The results illustrated that the infarct volume, brain water content and neurofunctional deficiency were significantly reduced by 7 days of breviscapine treatment in MCAO + Bre group, compared with those in MCAO + saline group. Meanwhile, the western blotting, quantitative PCR and immunofluorescence showed that the autophagy in both neurons and astrocytes at the penumbra were markedly attenuated by breviscapine admininstration. Moreover, these pharmacological effects of breviscapine could be counteracted by autophagy inducer Tat-Beclin-1. Our study suggests that breviscapine can provide a neuroprotection against transient focal cerebral ischemia, and this biological function is associated with attenuating autophagy in both neurons and astrocytes. © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction Cerebral stroke, especially ischemic stroke, is a major cause of permanent disability and death worldwide [1]. During ischemia, cells in the infarct core are suffered with necrosis and die within several minutes after stroke. Whereas in the surrounding region (the penumbra area), death spreads slowly for hours to days postinsult, thus, there is a precious time window to rescue injured cells, especially autophagic cells [2]. In fact, with an appropriate treatment, some cells with autophagy can survive [3]. Therefore, the autophagic cells in the ischemic penumbra are the attractive targets for stroke treatment.

* Corresponding author. E-mail address: [email protected] (D. Yihao). http://dx.doi.org/10.1016/j.biopha.2017.03.039 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

There are three types of cell death are caused by ischemic stroke: necrosis, apoptosis and autophagy. Necrosis is the end result of a bioenergetic catastrophe caused by ATP depletion to a level being incompatible with cell survival, and is thought to be an irreversible cell death. Apoptosis plays a modest protection in the cerebral ischemic stroke by delaying cell death via minimizing damage and disruption to neighboring cells [4], but an activated apoptosis pathway leads invariably to cell death. Autophagy is a cellular self-digestion process by depleting cellular components for cell survival, and is extensively involved in cerebral ischemia. Autophagy is generally considered to be a protective mechanism against ischemia, stress, starvation, and neurodegeneration [5–7]. However, autophagy is a double-edged sword for neuronal survival in cerebral ischemia [8]. Appropriate autophagic activity is neuroprotective [9], but excessive autophagy often leads to cell death [10]. Recent study found that both autophagy and apoptosis mechanisms were simultaneously activated by cerebral ischemia,

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and approximately 30% of cells in the ischemic penumbra presented both up-regulation of Beclin-1 (an autophagy initiator) and activation of caspase-3 (an effector protein of apoptosis), and the study further deduced that there might be intensive interactions between autophagy and apoptosis following cerebral stroke [11]. A study even revealed that autophagy preceded apoptosis and might initiate apoptosis [12]. These outcomes highlight the importance of autophagy in the pathological process of cerebral ischemia. Although the roles that autophagy played in cerebral ischemia are extensively controversial, most investigators believe that targeting autophagic pathway may find more insights into stroke treatment [13]. Recently, increasing evidence revealed that a neuroprotection against cerebral stroke could be gained by regulating autophagic activity [14,15]. Therefore, we especially focused on the pharmacological effect of breviscapine on autophagy in this study. Breviscapine is a flavonoid extracted from a traditional Chinese herb Erigerin breviscapus (Vant.) Hand-Mazz. The major active component of breviscapine is scutellarin (40 , 5, 6-tetrahydroxyflavone-7-O-glucuronide), which is able to improve microcirculation, promote cerebral blood flow [16], diminish aggregation of platelets and prevent oxidant [17]. In the light of these pharmacological activities, an injection preparation of breviscapine (a traditional Chinese patent medicine, TCPM) has been wildly used in clinical treatment for cerebral stroke in China [18]. Whereas its underlying pharmacological mechanisms are still not understood. The prominent pathological outcomes caused by cerebral ischemia are neuronal damage and neuronal death, hence, the past pharmaceutic clinical trials and basic research mostly focused on reducing neuronal injury and promoting neuronal survival. However, the intervention only targeting neuronal survival had ultimately failed to promote neurological recovery or reduce ischemia-induced injury [19]. The subsequent studies found that the survival and functional maintenance of glial cells, especially astrocytes, were required for neuronal survival after cerebral stroke [20]. In fact, astrocytes have a number of activities, such as cerebral microcirculation regulation, secretion of neuroprotective factors, glutamate homeostasis, maintenance of ion homeostasis and water balance [21,22], etc. Therefore, the contributions of astrocytes to neurofunctional rehabilitation after stroke have to be considered in the study of pharmacological mechanisms. In present study, we were to investigate whether breviscapine could augment a neuroprotection against cerebral ischemia injury, and further observed whether breviscapine targeted autophagy pathway to execute its pharmacological efficacy. 2. Materials and methods 2.1. Preparation of MCAO rat models and treatments All animal experiments were approved by the animal experiment committee of Kunming University of Science and Technology (approval number: SYXK K2013-0003, Kunming, China). Pathogen-free male Sprague-Dawley rats weighing 250–280 g were provided by the laboratory animal center of Kunming University of Science and Technology (Kunming, China). The animals were managed according to animal welfare practices. All surgery was performed under anesthesia with 10% chloral hydrate (360 mg/kg) by intraperitoneal injection (i.p.) for minimizing suffering. After anesthesia, the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) on the left were separated away from adjacent muscles and nerves, respectively. A 4-0 nylon monofilament coated with a round polylysine tip (diameter 0.36 mm, Beijing Shadong Biotechnology

Co., Ltd, Beijing, China) was inserted from a mini-incision on the ECA and went back into CCA, and then gently to middle cerebral artery (MCA) from ICA. During surgery, the blood pressure, arterial blood gases and heart rate were monitored. The nylon monofilament was withdrawn to allow reperfusion after 90 min of the middle cerebral artery occlusion (MCAO). In order to guarantee the robustness of the experimental stroke, a laser Doppler flowmetry (PeriFlux 5000, Perimed, Sweden) was used to monitor the regional cerebral blood flow during surgeries and after reperfusion. After the surgeries, the animals were randomly divided into 5 groups: Sham group (n = 13), the rats received the same surgery except for the insertion with a nylon monofilament and treated with neither breviscapine nor physiological saline; MCAO + Bre group (n = 16), the animals were treated with breviscapine injection (i.p., 0.33 mg/kg, administration dose in rats was determined by calculating an equivalent dose per unit weight to that used in clinical patients, Chinese national medicine permission number: Z53020707, Yunnan Yuyao Biological Pharmaceutical Co., Ltd, Yuxi, China) once daily for 7 days after onset of reperfusion; MCAO + saline group (n = 14), the rats were administrated with the same volume of physiological saline; MCAO + Tat-Beclin-1 group (n = 12), the animals were treated with Tat-Beclin-1 (Millipore, Billerica, MA, USA, 1.5 mg/kg, i.p.) at 3 and 6 days after MCAO; MCAO + Bre + Tat-Beclin-1 group (n = 14), the rats were delivered with Tat-Beclin-1 at 3 and 6 days during breviscapine treatment. 2.2. Neurological deficit score To assess the efficacy of breviscapine on neurofunctional deficiency caused by cerebral ischemia, the neurological deficit score was assessed after 7 days of breviscapine treatment following the MCAO/reperfusion. The neurological deficit score was determined by the following categories: 0, no observable neurological deficit; 1, flexion in the right forelimb; 2, failure to extend right forelimb completely and the strength to resist lateral push decreased obviously; 3, forelimb flexion, rotating and crawling towards right side; 4, unable or difficult to ambulate spontaneously. 2.3. Measurement of brain infarct volume After neurological deficit score, the rats were sacrificed after deep anaesthesia with 10% chloral hydrate (i.p. 400 mg/kg). The brains were quickly removed on ice and immediately frozen at 20  C for 20 min, and were then sliced into 2 mm thickness of sections. The brain sections were immediately stained with 0.5% triphenyltetrazolium chloride (TTC) solution (Shanghai haling Biotechnology Co., Ltd, Shanghai, China) at 37  C for 30 min, the sections were then fixed with 4% paraformaldehyde buffer (Shanghai Sangon Biotechnology Co., Ltd, Shanghai, China) for 12 h at room temperature. The infarct brain tissues were shown pale and unstained brain tissues were shown red by TTC staining. The infarction volume was calculated by imaging software (Adobe Photoshop 7.0), and was determined by infarction rate (%) = A /A'  100%, A represented the infarct volume, A' was the volume of the homolateral hemisphere. 2.4. Detection of brain water content To investigate the effect of breviscapine on cerebral edema caused by the MCAO/reperfusion, the water content in brain tissues were assessed by comparing dry weight with wet weight. The animals from each group were sacrificed after deep anaesthesia with 10% chloral hydrate (i.p. 400 mg/kg), the brains were soon removed and weighed for wet weight. The brain tissues

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were then dried off in an oven at 105  C overnight for dry weight. The water content was calculated by rate of water content (%) = (wet weight  dry weight)/wet weight  100%. 2.5. Protein isolation and western blotting Animals were sacrificed 7 days after the insult. The infarct penumbra and the corresponding area in the heterolateral brain tissues were isolated on ice. After homogenization by abrasiveness, the brain tissues were dealt with RIPA buffer (containing 1% Triton X-100, 0.1% SDS, 50 mM Tris pH 7.4, 1% sodium deoxycholate and 150 mM NaCl, Beijing BLKW Biotechnology Co., Ltd, Beijing, China) for 45 min at 4  C. Supernatants were collected by 13,000 g centrifugation for 15 min at 4  C. The proteins in the supernatants were first separated by SDS-PAGE gel electrophoresis and were then transferred into polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 10% nonfat milk for 2 h at room temperature under shaking, and were washed with PBST (PBS containing 0.1% polysorbate 20), and were then labeled with monoclonal rabbit antibodies against rat LC3 (Cell Signaling Technology, Danvers, MA, USA, 1:1000). After washing with PBST, the membrances were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Beijing Tiangen Bio-Technology, Beijing, China) for 1 h at room temperature. After 2 h washing with PBST, the reaction was visualized by electrochemiluminescence (ECL). The protein bands were measured in a Chemiluminescent reagent (Millipore, Billerica, MA, USA). The results were represented as fluorescence density ratio of LC3/beta actin.

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Technology, Danvers, MA, USA, 1:400), NeuN (abcam, Cambridge, UK, 1:300), mouse antibody against rat GFAP (Cell Signaling Technology, Danvers, MA, USA, 1:200) and PBS (negative control) for 4 h at room temperature. After a washing step, the sections were incubated with Alexa Fluor 594-conjugated anti-rabbit IgG (Invitrogen, Shanghai, China, 1:800) and Dylight 488-conjugated anti-mouse IgG (Invitrogen, Shanghai, China, 1:800) for 2 h in the dark, respectively. The sections were then counterstained with 40 , 6-diamidino-2-phenylindole (DAPI, Invitrogen, Shanghai, China) in PBS (1:1000) for 5 min. After washing, the reaction were detected by a fluorescentmicroscope (Nikon Instruments Co., Ltd., Tokyo to, Japan). The results were represented as percentages of positive cells. Under high power microscopy (400), the number of positive cells and total number of cells in the tissues were counted in 10 randomly selected fields from each section, respectively, and five tissue sections were counted from each detected sample. All counting was manually performed by an investigator who was blinded to the treatment among groups. 2.8. Statistical analysis All data were presented as means  standard error of the mean (SEM). Statistical differences were valuated by one-way analysis of variance (ANOVA) followed by Dunett’s test. P < 0.05 was considered as statistically significant. 3. Results 3.1. Breviscapine significantly reduced neurological deficiency caused by MCAO

2.6. Real-time PCR to measure LC3 mRNA levels The total RNA of brain tissues in the ischemic penumbra was extracted by TRIzol reagent (Invitrogen, Shanghai, China). The first-strand of complementary DNA (cDNA) was synthesized using 1 mg of the total RNA according to the guideline provided by manufacturer (Fermantas MBI, Germany). Thereafter, real-time PCR was performed using SYBR Green I and the primers: LC3, 50 -CAT GGG CAC AGA TGA AGA CAC-30 and 50 - GCC AGA TGT TCA TCC ACT TTC-30 ; b-actin, 50 -TAA AGA CCT CTA TGC CAA CAC AAG T30 and 50 -CAC GAT GGA GGG GCC GGA CTC ATC-30 . For PCR amplification, a 20 ml of reaction system (containing sterile distilled water, 2 ml of qTaqpolymerase (Takara, Japan), 2 ml of diluted cDNA and 0.2 mmol/L of each primer) was prepared. After amplification, a Mastercycler1 ep realplex (Eppendorf, Germany) was used to detect the reaction. The melting curve was analyzed to confirm that only single PCR products were being amplified. The cycle threshold (Ct) value was normalized to the value of the housekeeping gene b-actin. The relative mRNA level of LC3 was calculated by the 2DDCt method.

In order to investigate whether breviscapine could alleviate neurofunctional deficiency caused by ischemic stroke, the neurological deficit score was assessed after 7 days of breviscapine treatment. The result (Fig. 1) illustrated that there was obvious neurological deficiency was displayed in MCAO + saline group,

2.7. Detection of immunofluorescence After deep anaesthesia with 10% chloral hydrate (i.p., 400 mg/kg), the animals were fixed by transcardial perfusion with physiological saline followed by 4% paraformaldehyde (Invitrogen, Shanghai, China) after 7 days of breviscapine treatment following MCAO/reperfusion. The brains were removed and dehydrated with a 20% sucrose solution overnight (Invitrogen, Shanghai, China). The brain tissues were then sliced into sections (20 mm of thickness) with a freezing microtome (SLEE, Mainz, Germany). The brain sections were washed with PBS and permeabilized with 0.2% Triton X-100 in PBS at room temperature for 15 min, after washing with PBS, sections were blocked with 10% normal goat serum for 40 min. The sections were then respectively labeled with monoclonal rabbit antibodies against rat LC3 (Cell Signaling

Fig. 1. Neurological deficit score. In order to investigate whether breviscapine could provide a protective efficacy against neurofunctional deficiency caused by ischemic stroke, the neurological deficit score was assessed after 7 days of breviscapine treatment following the insult. The result illustrated that the neurological deficit score in MCAO + Bre group (n = 16) was significantly reduced by breviscapine treatment, and markedly lower than that in MCAO + saline group (n = 14). However, the effect of breviscapine to attenuate neurological deficiency was canceled by autophagy inducer Tat-Beclin-1 in MCAO + Bre + Tat-Beclin-1 group (n = 14). The neurological deficit score in MCAO + Tat-Beclin-1 group (n = 12) was obviously increased compared with that in MCAO + saline group, indicating that increased autophagy leads to neurofunctional damage. * P < 0.01.

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MCAO + Tat-Beclin-1 group and MCAO + Bre + Tat-Beclin-1 group. Comparatively, the neurological deficit score was significantly reduced by 7 days of breviscapine treatment in MCAO + Bre group (P < 0.01). 3.2. Breviscapine prominently decreased the infarct volume caused by MCAO To observe whether breviscapine could reduce brain damage caused by the MCAO, the cerebral infarct volume was measured by TTC staining. The result (Fig. 2) showed that the infarction size was obviously reduced by breviscapine treatment in MCAO + Bre group, compared with that in MCAO + saline group (P < 0.01). Comparatively, the infarct volume in MCAO + saline group, MCAO + TatBeclin-1 group and MCAO + Bre + Tat-Beclin-1 group was only mildly decreased 7 days after the insult. 3.3. Breviscapine markedly alleviated cerebral edema induced by MCAO/reperfusion In order to investigate whether breviscapine was able to diminish cerebral edema induced by the MCAO/reperfusion, the water content in the brain tissues was assessed. After 7 days of breviscapine treatment, the brain water content in MCAO + Bre group was obviously lower than that in MCAO + saline group, MCAO + Tat-Beclin-1 group or in MCAO + Bre + Tat-Beclin-1 group (P < 0.01, Fig. 3).

Fig. 3. Effect of breviscapine on brain edema caused by MCAO/reperfusion. After 7 days of breviscapine treatment following the insult, the brain water content in MCAO + Bre group was still higher than that in Sham group, but significantly lower than that in MCAO + saline group, or in MCAO + Tat-Beclin-1 group, indicating that breviscapine has an activity to alleviate ischemia-induced cerebral edema. However, this pharmacological effect was counteracted by autophagy inducer Tat-Beclin-1 in MCAO + Bre + Tat-Beclin-1 group. *P < 0.01.

LC3-II/LC3-I ratio was also significantly decreased by breviscapine treatment.

3.4. Breviscapine inhibited autophagic activity in the ischemic penumbra

3.5. Breviscapin decreased LC3 mRNA levels in the penumbra tissues

In order to investigate whether breviscapine could reduce autophagy induced by the MCAO, the autophagy levels were evaluated by western blotting. The result illustrated that the LC3 expression level in the penumbra was obviously reduced by 7 days of breviscapine treatment in MCAO + Bre group (Fig. 4), compared with that in MCAO + saline group, MCAO + Tat-Beclin-1 group or in MCAO + Bre + Tat-Beclin-1 group (P < 0.01), meanwhile, the

To further verify the efficacy of breviscapine to attenuate ischemia-induced autophagy, the LC3 mRNA level was evaluated by real-time PCR. The result (Fig. 5) indicated that LC3 transcription level was obviously reduced by 7 days of breviscapine treatment in MCAO + Pue group, compared with that in MCAO + saline group (P < 0.01). However, this effect of breviscapine to inhibit autophagy could be canceled by autophagy inducer

Fig. 2. Effect of breviscapine on cerebral infarct volume in MCAO brains. The infarct volume was detected after 7 days of breviscapine treatment. The normal tissue was red and the infarct tissue showed pale by TTC staining (A). There was no infarction observed in the Sham group. After 7 days of breviscapine treatment, the infarct volume in MCAO + Bre group was markedly reduced, and obviously lower than that in MCAO + saline group, or in MCAO + Bre + Tat-Beclin-1 group. However, after Tat-Beclin-1 administration, the infarct volume in MCAO + Tat-Beclin-1 group was even higher than that in MCAO + saline group (B), indicating that induction of autophagy leads to brain damage. *P < 0.01.

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Fig. 4. Efficacy of breviscapine on ischemia-induced autophgy in the penumbra. The autophagy in the ischemic penumbra was evaluated by western blotting with antibody LC3 (an autophagy indicator) 7 days after the insult (A). The results (B) illustrated that LC3-II expression level was significantly reduced by 7 days of breviscapine treatment in MCAO + Bre group, compared with that in MCAO + saline group, MCAO + Bre + Tat-Beclin-1 group or in MCAO + Tat-Beclin-1 group. The LC3-II/LC3-I ratio was also decreased in MCAO + Bre group (C), indicating autophagosome formation was markedly suppressed by breviscapine treatment. However, the efficacy of breviscapine to inhibit autophagy could be counteracted by autophagy inducer Tat-Beclin-1 in MCAO + Bre + Tat-Beclin-1 group. As we predicted, the autophagy level was obviously increased by Tat-Beclin-1 in MCAO + Tat-Beclin-1 group. *P < 0.01.

Tat-Beclin-1 in MCAO + Bre + Tat-Beclin-1 group. Expectantly, the LC3 mRNA level in MCAO + Tat-Beclin-1 group was significantly promoted by autophagy inducer of Tat-Beclin-1.

3.6. Autophagy both in nerons and astrocytes in the ischemic penumbra was markedly attenuated by breviscapine The effect of breviscapine to inhibit ischemia-induced autophagy was further proved by double immunofluorescence with antibodies of LC3, GFAP (an indicator of astrocytes) and NeuN (a marker of neurons), respectively (Fig. 6A, B). The result showed that the percentage of LC3-positive neurons (Fig. 6C) in MCAO + Bre group was significantly lower than that in MCAO + saline group, MCAO + Tat-Beclin-1 group or in MCAO + Bre + Tat-Beclin-1 group (P < 0.01). Meanwhile, the ratio of LC3-positive astrocytes was also markedly reduced by 7 days of breviscapine treatment in MCAO + Bre group (Fig. 6D). 4. Discussion

Fig. 5. Real-time PCR to detect LC3 mRNA levels in the brain tissues from the ischemic penumbra. After 7 days of breviscapine treatment, the LC3 mRNA transcription level was significantly reduced in MCAO + Bre group, compared with that in MCAO + saline group. However, this efficacy of breviscapine was counteracted by autophagy inducer Tat-Beclin-1 in the MCAO + Bre + Tat-Beclin-1 group. Contrarily, the LC3 mRNA was obviously increased by Tat-Beclin-1 administration in the MCAO + Tat-Beclin-1, compared with that in MCAO + saline group. *P < 0.01.

There is complex interaction among autophagy, apoptosis and necrosis [23]. In physiological situations, autophagy maintain cell survival by providing cytoplasmic components to support ATP production [8]. However, in the conditions of ischemia, both autophagy and apoptosis are simultaneously activated [24]. Undoubtedly, an activated apoptosis pathway leads invariably to cell death, but autophagy may either promote cell survival or mediate cell death. The roles that autophagy played in cerebral stroke are determined by the severity of ischemia [25]. On the one hand, a modest autophagy plays a survival role by providing energy to support apoptosis, a programmed death that requires energy to be executed, and by delaying necrosis. Meanwhile, the cells with autophagosomes can also produce energy again and survive if ischemia is attenuated [23]. On the other hand, in conditions of severe brain ischemia, over-activated autophagic cells may not have the required energy to survive, or to conclude the apoptotic

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Fig. 6. Immunofluorescence staining for LC3 expression in neurons and astrocytes in the ischemic penumbra. To investigate autophagy levels in both neurons and astrocytes in the ischemic penumbra, double immunofluorescence was performed with antibodies of LC3 (an autophagy indicator), NeuN (a neuron marker) and GFAP (an astrocyte marker), respectively. The results showed that the percentages both of LC3-positive neurons (C) and LC3-positive astrocytes (D) were obviously decreased by 7 days of breviscapine treatment in the MCAO + Bre group, compared with those in MCAO + saline group, MCAO + Bre + Tat-Beclin-1 group or in MCAO + Tat-Beclin-1. Being similar to the result of the western blotting, the efficacy of breviscapine to reduce autophagy could be abolished by autophagy inducer Tat-Beclin-1 in the MCAO + Bre + Tat-Beclin-1 group. A and B respectively illustrated the LC3-positive (red) neurons (green) and astrocytes (green), the blue shows DAPI staining, and the arrows indicate LC3-positive neurons or astrocytes (yellow). Bar = 10 mm. The black squares in Fig.E show the selected fields for counting of LC3-positive neurons or astrocytes. *P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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process, thus, the injured cells may progress towards necrosis [26]. In fact, there is a switch from autophagy to necrotic cell death, and this switch can be interrupted by neuroagents intervention [27]. A study have revealed that a neuroprotection against cerebral ischemia injury can be obtained by reversing autophagic activity [28]. The acute cerebral ischemia often induces excessive autophagy activity, and the over-activated autophagy may lead to cell death [29]. To date, it is difficult to define the line between appropriate and excessive is now. However, increasing study indicated that inhibition of autophagy was neuroprotective after stroke, such as exercise [30], exercise pretreatment [31], ischemic postcondition [32] and some drugs [33]. Therefore, it is rational to conduct that attenuating excessive autophagy can promote cell survival after cerebral stroke. Light chain 3 (LC3) localizes autophagosomal membranes after post-translational modifications, and is a homologue of Aut7/Apg8p being essential for autophagy in yeast. LC3 is first cleaved at the carboxy terminus immediately following synthesis to yield a cytosolic form of LC3-I. During autophagy, LC3-I is converted to an autophagosomeassociated form of LC3-II which represents the extent of autophagosome formation [34]. LC3 often cross-recognizes LC3-I and LC3-II, thus, both initial activated autophagy (LC3-I) and formed autophagy (LC3-II) can be detected by a LC3 antibody [35], and the LC3-II/LC3-I ratio reflects the activation level of autophagy. Therefore, the LC3 was used to evaluate autophagy levels in present study. Neuronal damage and its mechanisms involved in ischemic stroke have been extensively studied. However, recent evidence suggested that glial cells, especially astrocytes, also play unique roles in protecting brain function [36]. The importance of astrocytes to maintain homeostasis, and to provide extracellular microenvironment for neuronal survival are consequently received much attention in the study of cerebral stroke. During ischemia, astrocytes are able to survive even when neurons die in their surroundings and display adaptive changes to prevent further injury in neurons [37]. During recovery following ischemic insult, astrocytes store glycogen and provide lactate as an alternative aerobic energy substrate for neurons [38]. Additionally, astrocytes contribute to neuroprotective efficacy by producing various growth factors and cytokines as mediators of inflammation and immune responses [12]. Thus, astrocytes not only support themselves and protect other cells with which they were in intimate contact, but also actively take part in the demise of brain tissue after ischemia. Besides induction of neuronal autophagy, cerebral ischemia also resulted in excessive astrocytic autophagy which in turn led to cell death [39]. Therefore, in present study, we investigated efficacy of breviscapine on autophagy in both neurons and astrocytes, to reveal its neuroprotective mechanism. The results illustrated that the cerebral infarct volume and neurofunctional deficiency were significantly reduced by 7 days of breviscapine treatment in MCAO + Bre group, compared with those in MCAO + saline group (non-administration with breviscapine), indicating that breviscapine reliably has a neuroprotective efficacy against cerebral ischemia injury. This result is consistent with the previous study, which suggested that breviscapine was able to confer a neuroprotection against cerebral ischemia/reperfusion injury [40]. Meanwhile, the water content in the brain tissues was markedly decreased by breviscapine treatment. This result suggests that the neuroprotection conferred by breviscapine may be associated with alleviating cerebral ischemic edema. Astrocytes, neurons, microglia and oligodendrocytes can all produce inflammatory cytokines to sustain normal function of the central nervous system [41], but under conditions of ischemic stroke, excessive inflammatory responses often lead to neurofunctional pathology [42]. In our study, the water content in brain tissues were obviously declined by breviscapine treatment,

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indicating that breviscapine may be able to inhibit excessive inflammatory responses induced by the insult. In order to investigate the pharmacological mechanisms of breviscapine, we further evaluated the autophagy levels in the infarct penumbra by western blotting, real-time PCR and immunofluorescence, respectively. The results showed that LC3 expression level in MCAO + Bre group was significantly lower than that in MCAO + saline group. Comparatively, the autophagy still maintained in a high level in MCAO + saline group 7 days after the insult. Meanwhile, the LC3-II/LC3-I ratio was significantly decreased in MCAO + Bre group, indicating that the over-activated autophagy activity induced by MCAO/reperfusion could be efficiently inhibited by breviscapine. The double immunofluorescence illustrated that the LC3 expression levels in neurons and astrocytes was simultaneously reduced by breviscapine treatment, indicating that the neuroprotective mechanism of breviscapine was associated with autophagy suppression in both neurons and astrocytes. This result is also partly supported by the reports [33,43], which illustrated that inhibition of autophagy could protect brain from ischemic injury. Tat-Beclin-1, a specific autophagy inducer, was able to efficiently initiate autophagy by interacting with Beclin-1 [44], which was a key recruitment protein in the autophagic process [45]. In order to verify the specific efficacy of breviscapine to ameliorate ischemia-induced autophagy, the Tat-Beclin-1 was administrated at 3 and 6 days after breviscapine treatment. The result showed that the biological function of breviscapine could be abolished by Tat-Beclin-1, illustrating that the neuroprotective effects of breviscapine was specifically associated with inhibiting autophagic activity. In conclusion, our study showed that breviscapine could augment a prominent protection against transient focal cerebral ischemia, through inhibiting infarct expansion, reducing neurofunctional deficiency and alleviating cerebral edema. We further revealed that breviscapine was able to significantly reduce the ischemia-induced autophagy in neruons and astrocytes in the penumaba area. However, the neuroprotective function of breviscapine could be blocked by autophagy inducer of Tat-Beclin-1. So we conclude that breviscapine is able to provide a neuroprotection aganist cerebral ischemia, and this biological efficacy is associated with attenuating autophagy in both neurons and astrocytes in the penumbra. Conflicts of interest The authors declare no conflict of interest. Acknowledgement This study was funded by Chinese National Natural Science Foundation (No. 81660383 and No. 81460351). References [1] S.P. Klowka, M. Wintermark, T. Engelhorn, J.B. Fiebach, Acute stroke magnetic resonance imaging: current status and future perspective, Neuroradiology 52 (2010) 189–201. [2] T.S. Olsen, B. Larsen, M. Herning, E.B. Skriver, N.A. Lassen, Blood flow and vascular reactivity in collaterally perfused brain tissue: evidence of an ischemic penumbra in patients with acute stroke, Stroke 14 (1983) 332–341. [3] E.H. Lo, A new penumbra: transitioning from injury into repair after stroke, Nat. Med. 14 (2008) 497–500. [4] A.L. Edinger, C.B. Thompson, ;1; Death by design: apoptosis, necrosis and autophagy, Curr. Opin. Cell Biol. 16 (2004) 663–669. [5] R.A. Nixon, Autophagy in neurodegenerative disease: friend foe or turncoat? Trends Neurosci. 29 (2006) 528–535. [6] R.A. Nixon, The role of autophagy in neurodegenerative disease, Nat. Med. 19 (2013) 983–997. [7] Y.C. Wong, E.L. Holzbaur, Autophagosome dynamics in neurodegeneration at a glance, J. Cell Sci. 128 (2015) 1259–1267.

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