brain research 1558 (2014) 100–108
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Research Report
w007B protects brain against ischemia–reperfusion injury in rats through inhibiting inflammation, apoptosis and autophagy Qixin Bua, Xiaoyan Liua, Yuanjun Zhua, Ye Liub, Yinye Wanga,n a
Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China b Beijing Honghui New Medical Technology Co. Ltd., Beijing Daxing Biological Medicine Industry Base, Beijing 102600, China
ar t ic l e in f o
abs tra ct
Article history:
This study was designed to investigate the effect of w007B, a newly synthesized derivative of
Accepted 18 February 2014
honokiol, on MCAO reperfusion, and its therapeutic time window and related mechanisms in
Available online 5 March 2014
rats. Neurological deficit scores, infarct size and brain water content were measured after 24 h
Keywords:
reperfusion following 2 h ischemia. The results showed that w007B (10 and 50 μg/kg, IV
Cerebral ischemia–reperfusion
immediately after reperfusion) markedly decreased neurological deficit scores, reduced infarct
w007B
size and alleviated brain water content, and then 50 μg/kg w007B given within 3 h after
Therapeutic time window
reperfusion (5 h after ischemia) significantly attenuated ischemia-induced brain injury.
Apoptosis
Additionally, no sign of toxicity was observed when a single dose of 50 mg/kg w007B (1000
Inflammation
times of the highest effective dose, IP) was administered. To explore the underlying
Autophagy
mechanisms, the expression level of apoptosis, inflammation and autophagy-related markers in brain tissue were detected with kits or by western blot. It was observed that w007B rapidly and significantly reduced caspase-3 activity and NO production in the injured semi-brain, and also lowered the level of the p65 subunit of NF-κB in the nucleus. Besides, it also reduced the expression of Beclin-1 and LC3B-II, and increased the level of p62, the autophagy-related proteins in I/R-injured hemisphere. In conclusion, w007B exerts neuroprotective effect on cerebral ischemia–reperfusion injury with wider therapeutic time window and better safety; its mechanisms may be associated with its anti-inflammation, anti-apoptosis and antiautophagy action. These results suggest that w007B shows strong potential as a clinical neuroprotective candidate for the treatment of ischemic stroke. & 2014 Elsevier B.V. All rights reserved.
1.
Introduction
Ischemic stroke is one of the leading causes of mortality and disability with documented high incidence and relapse rate n
Corresponding author. Fax: þ86 10 62015584. E-mail address: [email protected] (Y. Wang).
http://dx.doi.org/10.1016/j.brainres.2014.02.034 0006-8993 & 2014 Elsevier B.V. All rights reserved.
(Roger et al., 2012). Although some effective thrombolytic agents have been applied in the treatment of acute ischemic stroke (Blakeley and Llinas, 2007), many victims without effective treatment suffer serious physical and cognitive
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disabilities, due to a further progression of neuronal damage after Ischemia–Reperfusion (I/R) (Goldstein and Rothwell, 2008). Therefore, neuroprotective therapy during ischemia and reperfusion is considered as a crucial strategy to reduce neuronal damage. Numerous clinical trials of neuroprotective agents in acute stroke have failed after promising results in animal models (Lo, 2009) and the need for clinically effective neuroprotective drugs is still very strong. Honokiol is the main biphenyl neolignan isolated from the cortex of Magnolia officinalis used in traditional Chinese medicine (Peng et al., 2010). Honokiol shows neuroprotective effects through various mechanisms, including anti-apoptosis (Yang et al., 2013), anti-inflammation (Zhang et al., 2013), antioxidation (Hu et al., 2006), anti-platelet aggregation (Pyo et al., 2002) and disturbing PSD95–NO interaction (Hu et al., 2013). However, its poor solubility and narrow therapeutic index restrict its application (Wang et al., 2011). w007B (Beijing Honghui New Medical Technology Co. Ltd., 2013), a newly synthesized derivative of honokiol, has a greatly improved water-solubility. This study aims at investigating the effects of w007B on cerebral I/R, its acute toxicity and main mechanisms of action.
the infarct volume to 31.073.6% and 12.172.4%, respectively (Fig. 2B). The inhibitory percent of infarct volume for 10 mg/kg and 50 mg/kg w007B was about 35.2% and 74.6%, respectively. Besides, w007B reduced the neurological deficit scores in a dose-dependent manner, consistent with the effect on the infarct volume (Fig. 2C). In addition, 10–50 mg/kg w007B significantly decreased brain water content (Fig. 2D). In order to assess the therapeutic time window of w007B, brain infarct volume, neurological deficit scores and brain water content in rats treated with w007B were measured 0, 1, 3 and 5 h after reperfusion (2, 3, 5, and 7 h after ischemia). Infarct volume was observed 24 h after reperfusion (Fig. 3A, white region), and w007B administered 0–3 h after reperfusion effectively reduced infarct volume (Fig. 3B). Inhibition of infarct volume at 0, 1 and 3 h was measured at 74.6%, 67.1% and 27.3%, respectively. w007B reduced the neurological deficit scores in a time-dependent manner (from 0 to 3 h after reperfusion or 2 to 5 h after ischemia) (Fig. 3C). In addition, w007B significantly decreased brain water content when administered 0 or 1 h after reperfusion (2 or 3 h after ischemia) (Fig. 3D).
2.3.
2.
Assessment of preliminary acute toxicity
Results
2.1. Cerebral blood flow (CBF) monitoring of LDF measurement Filament occlusion and laser-Doppler flowmetry (LDF) measurements were performed as described in Section 4. A minimum reduction of 70% of the initial LDF reading was considered as a successful occlusion of middle cerebral artery (MCA) (Fig. 1).
2.2.
101
w007B protected brain against I/R injury
To detect the effect of w007B against I/R injury, vehicle or agents were immediately administered after reperfusion (single IV bolus). In middle cerebral artery occlusion (MCAO) model group, the infarct volume was 47.872.8% (Fig. 2A, white region). The infarct volume for 2 mg/kg w007B was not significantly reduced. 10 mg/kg and 50 mg/kg w007B reduced
To determine the preliminary acute toxicity, w007B and its precursor honokiol (1000 50 mg/kg) or vehicles were administered to mice by a single dose of intraperitoneal injection (IP). No abnormal symptoms were observed for animals treated with w007B and vehicles. However, mice treated with honokiol showed depressive status, such as hypokinesia and lethargy, 60% of mice were dead within 6 d (Table 1). Body weights of mice treated with w007B and vehicles in the two weeks did not show significant variation (Fig. 4). These results suggest that w007B presents a lower toxicity than honokiol but further studies are needed to obtain a more complete safety assessment.
2.4.
w007B inhibited caspase-3 in I/R-injured hemisphere
In order to explore the underlying mechanisms of w007B's action, an investigation of its potential inhibition of cell apoptosis induced by ischemia–reperfusion injury was conducted. Caspase-3 activity in semi-brain tissue was detected by colorimetric assay. The caspase-3 relative activity was expressed as the fold of sham operation group, it was 3.597 0.4 and 1.8570.62 for model rats and for 50 mg/kg of w007Btreated rats, respectively. The results showed that caspase-3 relative activity was significantly decreased by w007B, indicating an inhibitory effect of w007B on apoptosis (Fig. 5).
2.5. w007B decreased NO production and nuclear p65 subunit of NF-κB in I/R-injured hemisphere
Fig. 1 – CBF monitoring during filament occlusion. The laser Doppler probe was located 4–5 mm lateral and 1–2 mm posterior to Bregma. PU: perfusion units.
The results showed that nitric oxide (NO) level was significantly increased in I/R semi-brain (0.6870.16 mM/mg tissue) compared with that in sham operation brain (0.1070.01 mM/mg tissue), and NO level in w007B-treated brain tissue was significantly reduced (0.1770.02 mM/mg tissue) (Fig. 6A), that is, w007B reduced NO production by 87.9%.
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Fig. 2 – The protective effect of w007B on cerebral I/R in rats. Vehicle or agents were administered at 0 h after reperfusion (IV). (A) Representation of TTC-strained brain slices at 24 h after reperfusion. (B) Infarct volume. (C) Neurological deficit scores. (D) Brain water content. Data were expressed as mean7SEM, n ¼ 10. 44 Po0.01 versus sham; nPo0.05, nnPo0.01 versus I/R.
Then the influence of w007B on NF-κB pathway activation in I/R semi-brain was examined. The results showed that the level of nucleus p65, one subunit of the NF-κB, was elevated in I/R-injured hemisphere to about 3.5 fold compared with ipsilateral hemisphere of the sham operation. w007B significantly reduced the elevated p65 expression (from 3.5070.99 to 1.4570.14) (Fig. 6B). These results suggest that w007B may also act on the signaling pathways related to inflammation.
2.6.
w007B inhibited autophagy in I/R-injured hemisphere
To investigate whether w007B can also inhibit the autophagy induced by I/R injury, we examined the expression level of several proteins related to autophagy. Concurrent increase of both Beclin-1 and LC3B-II and decrease of p62 represents activation of autophagy. The results showed that the relative expression of both Beclin-1 and LC3B-II were significantly increased in I/R semi-brain, and w007B significantly reduced the elevated expression of the two proteins (Fig. 7B and C) by 70.3% and 57.5%, respectively. Additionally, the relative
expression of p62 protein was decreased in ipsilateral hemisphere, and w007B significantly elevated the level of p62 compared with I/R model (Fig. 7D). These results imply that w007B may also confer the neuroprotective effect via inhibition of autophagy activated by I/R injury.
3.
Discussion
Honokiol is a compound with neuroprotective activity (Lin et al., 2006), but has some limits in application for clinical trials (Wang et al., 2011). w007B was synthesized as a honokiol precursor presenting a much higher water-solubility. This study demonstrates that its pharmacological effect on cerebral I/R injury in rats remains prominent. Our results showed that w007B markedly improved neurological deficit, reduced both infarct volume and brain edema when given by single IV bolus after reperfusion. We also simultaneously observed the effects of honokiol and three agents clinically applied in China (positive controls).
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Fig. 3 – The time window of w007B on cerebral I/R in rats. Vehicle or agents were administered at different time after reperfusion (IV). (A) Representation of TTC-strained brain slices at 24 h after reperfusion. (B) Infarct volume. (C) Neurological deficit scores. (D) Brain water content. Data were expressed as mean7SEM, n ¼ 10. 44 Po0.01 versus sham; nPo0.05, nnPo0.01 versus I/R. Table 1 – Signs caused by high dose of w007B and honokiol administrated (IP). Animal group
Agenta
Male
w007B NS Honokiol
Female
0 0 60
Solution A
0
w007B NS
0 0
Honokiol Solution A a
Mortality
60 0
Survival times (d)
Symptoms
– – 2 3 5 –
None None Motionless Motionless Motionless None
– – 3 5 6 –
None None Motionless Motionless Motionless None
The dose of both w007B and honokiol were 50 mg/kg.
w007B (50 μg/kg) reduced the infarct volume by 74.6%, which is similar to the results obtained with honokiol in the same mole dose, and the three positive agents (edaravone, 3 mg/kg; butylphthalide, 50 mg/kg; and nimodipine, 1 mg/kg) which reduced the infarct volume by 62.8%, 60.6% and 61.2% respectively. Besides, the effect on neurological deficit scores was similar to that on infarct volume (Fig. 2), suggesting that w007B
has more potent neuroprotective effect than these positive agents. In order to detect the therapeutic time window, w007B was administered at 0, 1, 3 and 5 h after reperfusion. The results showed that it significantly reduced infarct volume when given at 0–3 h after reperfusion (2–5 h after ischemia) (Fig. 3), suggesting that the therapeutic time window is at least 3 h after reperfusion or 5 h after ischemia.
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In order to evaluate the safety of w007B, mice were treated with a single dose w007B (1000 times the highest effective dose). No sign of toxicity or mortality was observed, in
Fig. 4 – The influence on average weight of mice in preliminary acute toxicity experiment. Data were expressed as mean7SEM, n ¼5.
Fig. 5 – The influence on caspase-3 activity in rat brain after cerebral I/R. Casepase-3 activity was expressed as the fold of sham group. Data were expressed as mean7SEM, n¼ 5. 44 Po0.01 versus sham; nnPo0.01 versus vehicle.
contrast with honokiol which, used in the same method, resulted in the death of 60% of the mice within 6 d. These findings strongly suggest that w007B may be safer than honokiol. Apoptosis has been implicated in cerebral ischemic injury (Charriaut-Marlangue et al., 1998) and honokiol has been proved to inhibit endothelial cell apoptosis (Zhang et al., 2007). As a honokiol derivative, w007B may influence the process of apoptosis. Some studies have shown that caspase3 acts as an effector caspase and determines the incidence of apoptosis (Du et al., 1997). In order to investigate the effect of w007B on apoptosis induced by I/R injury, the activity of caspase-3 was monitored. Results showed that w007B reduces caspase-3 activity in I/R-injured hemisphere by 67.2% (Fig. 5). This observation suggests that w007B may exert its neuroprotective effect through the inhibition of neuronal cells apoptosis. NO is an important cytotoxic factor in the process of inflammation (Liu et al., 2002). Our previous studies showed that honokiol inhibited NO production in gial cells induced by LPS (Zhang et al., 2013) and in cortical neurons induced by glutamate and glycine (Hu et al., 2013) and it was therefore supposed that w007B, as a honokiol derivative, may have a similar action mechanism. w007B was indeed found to markedly reduce NO level in I/R tissue (Fig. 6A), which suggests inhibition of NO production during I/R. In addition, NF-κB plays a key role in the initiation of inflammation (Hyun-Ja et al., 2010) and is involved in I/R injury (Khan et al., 2012). It has been demonstrated that NF-κB is activated after transient MCAO, as revealed by the nuclear translocation of the NF-κB subunit p65 (Chen et al., 2009). We observed that w007B inhibited the level of the p65 subunit of NF-κB in the nucleus by 82.0% (Fig. 6B). These data suggested the inhibition of w007B on inflammatory response may be one of crucial mechanisms against I/R injury. Autophagy has been demonstrated to be involved in cerebral injury induced by ischemic stroke (Adhami et al., 2006). Beclin-1 and LC3 have been reported as two reliable markers in the study of autophagy (Rami et al., 2008; Mizushima et al., 2010). LC3 is expressed as three isoforms in mammalian cells, LC3A, LC3B and LC3C, but only LC3B-II
Fig. 6 – The influence on inflammation in cerebral I/R. (A) Effect of w007B on NO release in cerebral hemisphere 24 h after reperfusion. (B) Western blot analysis for the expression of the p65 subunit of NF-κB in cerebral hemisphere and statistical analysis. Data were expressed as mean7SEM, n ¼5, 44 Po0.01 versus sham; nnPo0.01 versus vehicle.
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Fig. 7 – The influence on autophagy in rat brain after cerebral I/R. Western blot analysis for the expression of proteins associate with autophage in cerebral hemisphere 24 h after reperfusion (A). Statistical analysis of Beclin-1, LC3B-II and p62 expression (B, C and D). Data were expressed as mean7SEM, n ¼ 5. 44 Po0.01 versus sham; nPo0.05 versus vehicle.
correlates with increased levels of autophagic vesicles (Barth et al., 2010). In addition, the autophagy-related protein p62/ SQSTM1 is selectively incorporated into autophagosomes through direct binding to LC3, then degraded by autophagy, namely, the p62 protein is unable to be cyclically used (Komatsu et al., 2007). Monitoring of the levels of Beclin-1, LC3B-II and p62 revealed a decrease of both Beclin-1 and LC3B-II, and an increase of p62 levels in the w007B-treated tissue, compared with vehicle-treated tissue. These observations suggest that w007B may exert its inhibitory effect by inactivating autophagy through decreasing Beclin-1 and LC3B-II, and increasing the level of p62, and thus achieve a cerebral protective effect. In conclusion, this study provides some evidence that w007B, a newly synthesized derivative of honokiol, significantly protected rats against ischemia–reperfusion induced brain injury. Besides, w007B has a therapeutic time window of at least 3 h after reperfusion or 5 h after ischemia. Moreover, it is less toxic than its precursor honokiol. It may exert neuroprotective effect, at least partly, through inhibiting the processes of inflammation, apoptosis and autophagy in I/Rinjured brain. All data suggest that w007B can be further developed as a clinical neuroprotective candidate in ischemic stroke.
4.
Experimental procedures
4.1.
Chemicals and agents
w007B and honokiol were provided by Beijing Honghui New Medical Technology Co. Ltd. (China), edaravone injection (Edar) was obtained from Simcere (Nanjing, China), butylphthalide soft capsules (NBP) was obtained from ShiJiaZhuang Pharma Group NBP Pharmaceutical Co. Ltd. (China), nimodipine injection (Nim) was from Bayer Schering Pharma AG (Germany). Honokiol was dissolved in solution A which was composed of polyethylene glycol 400/ethanol/water [40%/10%/50% (v/v/v)].
4.2.
Animals
ICR mice (19–21 g) and male SD rats (240–270 g) were provided by Department of Laboratory Animal Science, Peking University Health Science Center. All experimental procedures were approved by the Beijing Committee on Animal Care and Use. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Peking University Health Science Center (Permit number: LA2013-69). All animals were kept on a 12-h light/12-h dark regime, with free access to food and water.
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4.3.
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MCAO reperfusion model in rats
Transient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) as described previously (Hu et al., 2013) under anesthesia with chloral hydrate (350 mg/kg, IP), the filament was removed 2 h after MCAO. Sham-operated control rats received the same procedure except filament induction. The body temperature of rats was maintained and monitored at 3770.5 1C by holding operation table, and relative regional Cerebral Blood Flow (rCBF) was monitored with laser Doppler measurement (PeriFlux System 5000) during the surgery. A minimum reduction of 70% of the initial reading of LDF was considered as a successful occlusion of MCA perfusion territory (DiNapoli et al., 2006). Experiment 1: Ninety rats were randomly assigned to nine groups: sham group, vehicle group (normal saline), w007Btreated groups in different doses (2, 10, 50 mg/kg), honokioltreated group (28.5 mg/kg), edaravone-treated group (3 mg/kg), NBP-treated group (50 mg/kg) and nimodipine-treated group (1 mg/kg), 10 rats per group. Vehicle or agents were administered immediately after reperfusion intravenously. Experiment 2: Sixty rats were randomly assigned to six groups: sham group, vehicle group (normal saline), w007Btreated groups at different time points (0, 1, 3, 5 h after reperfusion), 10 rats per group. Animals were treated at different times after reperfusion intravenously. Neurological deficit was evaluated 24 h after reperfusion according to the Longa's method (Longa et al., 1989). Then the brains were dissected and cut into 5 coronal slices, 2-mm each, the slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC, Amresco, USA) for 15 min at 37 1C, according to the previous method (Bederson et al., 1986) with slight modification, followed by immersion-fixation in 4% paraformaldehyde. TTC-stained sections were photographed and the digital images were analyzed using image analysis software (NIH ImageJ software). Infarct areas were first measured using image analysis software and then compiled to obtain the infarct volume (mm3) per brain. The lesion volumes were calculated as a percentage of the contralateral hemisphere volume to compensate for the effect of brain edema using the following formula (Tatlisumak et al., 1998): f½total infarct volume ðthe volume of intact ipsilateral hemisphere the volume of intact contralateral hemisphereÞ =contralateral hemisphere volumeg 100%:
Cerebral edema extent was determined by measuring the brain water content according to the wet–dry method as described in previous reports (Chao et al., 2010) with some modifications. In brief, brains were immediately weighed to obtain wet weight after removal, then dried in an oven at 110 1C for 16 h and weighed again to obtain the dry weight. Brain water content was calculated as follows: Brain water content ð%Þ ¼ ½ðwet weightdry weightÞ=wet weight 100%:
4.4.
Preliminary acute toxicity
Forty ICR mice were randomly assigned to four groups: w007B-treated group, vehicle control group (normal saline), honokiol-treated group, and vehicle control group of honokiol (solution A). Following a single dose of the agents (50 mg/kg; IP), mice were observed for 14 d. All the animals were weighed before the treatment and the following days. Mortality, survival times (days) and symptoms induced were recorded within 14 d.
4.5.
Determination of casepase-3 activity
The influence of w007B on I/R-induced apoptosis was evaluated by detecting the activity of caspase-3 using a Colorimetric Assay Kit (KeyGen Biotech, Nanjing, China). Proteins were extracted from fresh ischemia ipsilateral cortical samples 24 h after reperfusion. Tissues were homogenized in cold lysis buffer containing 25 mM HEPES, pH 7.5, 5 mM MgCl2, 2 mM EDTA, 0.1% Triton 100, 2 mM dithiothreitol and 1 mM PMSF. The homogenates were centrifuged at 12,000g for 30 min at 4 1C, and the supernatants were collected. The protein concentration was determined by BCA assay (M&C Gene Technology, Beijing, China) and adjusted to 10 mg/ml. Then 500 mg proteins were added to 50 ml 2 reaction buffer supplement with 5 ml caspase-3 substrate and incubated at 37 1C away from light for 4 h. Caspases-3 activities were quantified spectrophotometrically at a wavelength of 405 nm. The activity of caspase-3 was expressed as the fold of the activity of sham group.
4.6.
Determination of inflammatory markers
The influence of w007B on I/R-induced inflammatory molecule of NO was determined by 2,3-diaminonaphthalene (DAN; Fluka) as previously reported (Misko et al., 1993). In brief, cerebrum was collected and homogenized in cold PBS; the supernatant was obtained by centrifugation at 1000g for 10 min. All procedures were performed at 4 1C. Supernatants were either used directly or stored at 80 1C. Protein concentrations were determined by the BCA method, and 100 ml of the supernatants of brains tissue supernatants was transferred to 96-well plates, mixed immediately with the solution of DAN (20 μl; 50 μg/ml in 0.62 M HCl), and incubated for 15 min in the dark at 25 1C. The fluorescence was measured with a FlexStation3 microplate reader with excitation at 365 nm and emission at 450 nm after adding NaOH (100 ml; 0.28 M). The nitrite concentration was determined based on the standard regression line for NaNO2. The nuclear translocation level of p65 subunit of NF-κB was detected through western blotting, nucleus proteins extracted with cold Nuclear and Cytoplasmic Extraction Kit (CWBIO, Beijing, China) were analyzed by Western blot. Western blot procedure followed standard protocols (Shimamura et al., 2006). Shortly, 35 μg of total protein were separated in 12% SDSpolyacrylamide gel electrophoresis (PAGE) and blotted onto PVDF membranes (Millipore, Billerica, MA, USA). Antibodies included Mouse anti-NF-κB p65 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Histone H1 (1:500, Zhongshan Golden Bridge Bio-technology, Beijing, China) and appropriate secondary antibodies (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein bands were detected with
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a chemiluminescent reagent (Millipore, Billerica, MA, USA). Optical density of the bands was quantified in Image Lab software, and the housekeeping protein Histone H1 was used as a control and tested simultaneously by mouse monoclonal anti-Histone H1 antibody.
4.7.
Determination of autophagy-related proteins
The effect of w007B on I/R-induced autophagy by western blot analysis with autophagy specific antibodies against Beclin-1, LC3B-II and p62, proteins extracted from the brains in cold RIPA Lysis Buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% C24H40O4 Na, 0.1% SDS, 2 mM Na4P2O7, 25 mM β-glycerophosphate, 1 mM Na3VO4 and 1 mM PMSF) were subjected to Western blot analysis. Western blotting procedure followed standard protocols (Shimamura et al., 2006): 35 μg of total protein were separated in 15% or 12% SDS-PAGE and blotted onto PVDF membranes (Millipore, Billerica, MA, USA). Antibodies included rabbit anti-Beclin-1 antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-LC3B antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-p62 antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-β-actin (1:1000, Zhongshan Golden Bridge Bio-technology, Beijing, China), and appropriate secondary antibodies (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein bands were detected with a chemiluminescent reagent (Millipore, Billerica, MA, USA). Optical density of the bands was quantified in Image Lab software, and the housekeeping protein β-actin was used as a control and tested simultaneously by mouse monoclonal anti-β-actin antibody.
4.8.
Statistical analysis
All data were expressed as mean7SEM. The one way ANOVA was used to assess differences among groups, Significant differences between groups at each time point were assessed by Student's t-test. Po0.05 was considered to be statistically significant.
Acknowledgments This work was supported by the Chinese National Technology Graveness Special Purpose Fund (2009zx09102-146) and the National Natural Science Foundation of China (61071002).
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Pyo, M.K., Lee, Y., Yun-Choi, H.S., 2002. Anti-platelet effect of the constituents isolated from the barks and fruits of Magnolia obovata. Arch. Pharm. Res. 25 (3), 325–328. Rami, A., Langhagen, A., Steiger, S., 2008. Focal cerebral ischemia induces upregulation of Beclin 1 and autophagy-like cell death. Neurobiol. Dis. 29 (1), 132–141. Roger, V.L., Go, A.S., Lloyd-Jones, D.M., Benjamin, E.J., Berry, J.D., Borden, W.B., Turner, M.B., 2012. Executive summary: heart disease and stroke statistics – 2012 update: a report from the American Heart Association. Circulation 125 (1), 188. Shimamura, N., Matchett, G., Solaroglu, I., Tsubokawa, T., Ohkuma, H., Zhang, J., 2006. Inhibition of integrin αvβ3 reduces blood–brain barrier breakdown in focal ischemia in rats. J. Neurosci. Res. 84 (8), 1837–1847. Tatlisumak, T., Carano, R.A., Takano, K., Opgenorth, T.J., Sotak, C.H., Fisher, M., 1998. A novel endothelin antagonist, A-127722, attenuates ischemic lesion size in rats with temporary middle
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Research Report
w007B protects brain against ischemia–reperfusion injury in rats through inhibiting inflammation, apoptosis and autophagy Qixin Bua, Xiaoyan Liua, Yuanjun Zhua, Ye Liub, Yinye Wanga,n a
Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China b Beijing Honghui New Medical Technology Co. Ltd., Beijing Daxing Biological Medicine Industry Base, Beijing 102600, China
ar t ic l e in f o
abs tra ct
Article history:
This study was designed to investigate the effect of w007B, a newly synthesized derivative of
Accepted 18 February 2014
honokiol, on MCAO reperfusion, and its therapeutic time window and related mechanisms in
Available online 5 March 2014
rats. Neurological deficit scores, infarct size and brain water content were measured after 24 h
Keywords:
reperfusion following 2 h ischemia. The results showed that w007B (10 and 50 μg/kg, IV
Cerebral ischemia–reperfusion
immediately after reperfusion) markedly decreased neurological deficit scores, reduced infarct
w007B
size and alleviated brain water content, and then 50 μg/kg w007B given within 3 h after
Therapeutic time window
reperfusion (5 h after ischemia) significantly attenuated ischemia-induced brain injury.
Apoptosis
Additionally, no sign of toxicity was observed when a single dose of 50 mg/kg w007B (1000
Inflammation
times of the highest effective dose, IP) was administered. To explore the underlying
Autophagy
mechanisms, the expression level of apoptosis, inflammation and autophagy-related markers in brain tissue were detected with kits or by western blot. It was observed that w007B rapidly and significantly reduced caspase-3 activity and NO production in the injured semi-brain, and also lowered the level of the p65 subunit of NF-κB in the nucleus. Besides, it also reduced the expression of Beclin-1 and LC3B-II, and increased the level of p62, the autophagy-related proteins in I/R-injured hemisphere. In conclusion, w007B exerts neuroprotective effect on cerebral ischemia–reperfusion injury with wider therapeutic time window and better safety; its mechanisms may be associated with its anti-inflammation, anti-apoptosis and antiautophagy action. These results suggest that w007B shows strong potential as a clinical neuroprotective candidate for the treatment of ischemic stroke. & 2014 Elsevier B.V. All rights reserved.
1.
Introduction
Ischemic stroke is one of the leading causes of mortality and disability with documented high incidence and relapse rate n
Corresponding author. Fax: þ86 10 62015584. E-mail address: [email protected] (Y. Wang).
http://dx.doi.org/10.1016/j.brainres.2014.02.034 0006-8993 & 2014 Elsevier B.V. All rights reserved.
(Roger et al., 2012). Although some effective thrombolytic agents have been applied in the treatment of acute ischemic stroke (Blakeley and Llinas, 2007), many victims without effective treatment suffer serious physical and cognitive
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disabilities, due to a further progression of neuronal damage after Ischemia–Reperfusion (I/R) (Goldstein and Rothwell, 2008). Therefore, neuroprotective therapy during ischemia and reperfusion is considered as a crucial strategy to reduce neuronal damage. Numerous clinical trials of neuroprotective agents in acute stroke have failed after promising results in animal models (Lo, 2009) and the need for clinically effective neuroprotective drugs is still very strong. Honokiol is the main biphenyl neolignan isolated from the cortex of Magnolia officinalis used in traditional Chinese medicine (Peng et al., 2010). Honokiol shows neuroprotective effects through various mechanisms, including anti-apoptosis (Yang et al., 2013), anti-inflammation (Zhang et al., 2013), antioxidation (Hu et al., 2006), anti-platelet aggregation (Pyo et al., 2002) and disturbing PSD95–NO interaction (Hu et al., 2013). However, its poor solubility and narrow therapeutic index restrict its application (Wang et al., 2011). w007B (Beijing Honghui New Medical Technology Co. Ltd., 2013), a newly synthesized derivative of honokiol, has a greatly improved water-solubility. This study aims at investigating the effects of w007B on cerebral I/R, its acute toxicity and main mechanisms of action.
the infarct volume to 31.073.6% and 12.172.4%, respectively (Fig. 2B). The inhibitory percent of infarct volume for 10 mg/kg and 50 mg/kg w007B was about 35.2% and 74.6%, respectively. Besides, w007B reduced the neurological deficit scores in a dose-dependent manner, consistent with the effect on the infarct volume (Fig. 2C). In addition, 10–50 mg/kg w007B significantly decreased brain water content (Fig. 2D). In order to assess the therapeutic time window of w007B, brain infarct volume, neurological deficit scores and brain water content in rats treated with w007B were measured 0, 1, 3 and 5 h after reperfusion (2, 3, 5, and 7 h after ischemia). Infarct volume was observed 24 h after reperfusion (Fig. 3A, white region), and w007B administered 0–3 h after reperfusion effectively reduced infarct volume (Fig. 3B). Inhibition of infarct volume at 0, 1 and 3 h was measured at 74.6%, 67.1% and 27.3%, respectively. w007B reduced the neurological deficit scores in a time-dependent manner (from 0 to 3 h after reperfusion or 2 to 5 h after ischemia) (Fig. 3C). In addition, w007B significantly decreased brain water content when administered 0 or 1 h after reperfusion (2 or 3 h after ischemia) (Fig. 3D).
2.3.
2.
Assessment of preliminary acute toxicity
Results
2.1. Cerebral blood flow (CBF) monitoring of LDF measurement Filament occlusion and laser-Doppler flowmetry (LDF) measurements were performed as described in Section 4. A minimum reduction of 70% of the initial LDF reading was considered as a successful occlusion of middle cerebral artery (MCA) (Fig. 1).
2.2.
101
w007B protected brain against I/R injury
To detect the effect of w007B against I/R injury, vehicle or agents were immediately administered after reperfusion (single IV bolus). In middle cerebral artery occlusion (MCAO) model group, the infarct volume was 47.872.8% (Fig. 2A, white region). The infarct volume for 2 mg/kg w007B was not significantly reduced. 10 mg/kg and 50 mg/kg w007B reduced
To determine the preliminary acute toxicity, w007B and its precursor honokiol (1000 50 mg/kg) or vehicles were administered to mice by a single dose of intraperitoneal injection (IP). No abnormal symptoms were observed for animals treated with w007B and vehicles. However, mice treated with honokiol showed depressive status, such as hypokinesia and lethargy, 60% of mice were dead within 6 d (Table 1). Body weights of mice treated with w007B and vehicles in the two weeks did not show significant variation (Fig. 4). These results suggest that w007B presents a lower toxicity than honokiol but further studies are needed to obtain a more complete safety assessment.
2.4.
w007B inhibited caspase-3 in I/R-injured hemisphere
In order to explore the underlying mechanisms of w007B's action, an investigation of its potential inhibition of cell apoptosis induced by ischemia–reperfusion injury was conducted. Caspase-3 activity in semi-brain tissue was detected by colorimetric assay. The caspase-3 relative activity was expressed as the fold of sham operation group, it was 3.597 0.4 and 1.8570.62 for model rats and for 50 mg/kg of w007Btreated rats, respectively. The results showed that caspase-3 relative activity was significantly decreased by w007B, indicating an inhibitory effect of w007B on apoptosis (Fig. 5).
2.5. w007B decreased NO production and nuclear p65 subunit of NF-κB in I/R-injured hemisphere
Fig. 1 – CBF monitoring during filament occlusion. The laser Doppler probe was located 4–5 mm lateral and 1–2 mm posterior to Bregma. PU: perfusion units.
The results showed that nitric oxide (NO) level was significantly increased in I/R semi-brain (0.6870.16 mM/mg tissue) compared with that in sham operation brain (0.1070.01 mM/mg tissue), and NO level in w007B-treated brain tissue was significantly reduced (0.1770.02 mM/mg tissue) (Fig. 6A), that is, w007B reduced NO production by 87.9%.
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Fig. 2 – The protective effect of w007B on cerebral I/R in rats. Vehicle or agents were administered at 0 h after reperfusion (IV). (A) Representation of TTC-strained brain slices at 24 h after reperfusion. (B) Infarct volume. (C) Neurological deficit scores. (D) Brain water content. Data were expressed as mean7SEM, n ¼ 10. 44 Po0.01 versus sham; nPo0.05, nnPo0.01 versus I/R.
Then the influence of w007B on NF-κB pathway activation in I/R semi-brain was examined. The results showed that the level of nucleus p65, one subunit of the NF-κB, was elevated in I/R-injured hemisphere to about 3.5 fold compared with ipsilateral hemisphere of the sham operation. w007B significantly reduced the elevated p65 expression (from 3.5070.99 to 1.4570.14) (Fig. 6B). These results suggest that w007B may also act on the signaling pathways related to inflammation.
2.6.
w007B inhibited autophagy in I/R-injured hemisphere
To investigate whether w007B can also inhibit the autophagy induced by I/R injury, we examined the expression level of several proteins related to autophagy. Concurrent increase of both Beclin-1 and LC3B-II and decrease of p62 represents activation of autophagy. The results showed that the relative expression of both Beclin-1 and LC3B-II were significantly increased in I/R semi-brain, and w007B significantly reduced the elevated expression of the two proteins (Fig. 7B and C) by 70.3% and 57.5%, respectively. Additionally, the relative
expression of p62 protein was decreased in ipsilateral hemisphere, and w007B significantly elevated the level of p62 compared with I/R model (Fig. 7D). These results imply that w007B may also confer the neuroprotective effect via inhibition of autophagy activated by I/R injury.
3.
Discussion
Honokiol is a compound with neuroprotective activity (Lin et al., 2006), but has some limits in application for clinical trials (Wang et al., 2011). w007B was synthesized as a honokiol precursor presenting a much higher water-solubility. This study demonstrates that its pharmacological effect on cerebral I/R injury in rats remains prominent. Our results showed that w007B markedly improved neurological deficit, reduced both infarct volume and brain edema when given by single IV bolus after reperfusion. We also simultaneously observed the effects of honokiol and three agents clinically applied in China (positive controls).
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Fig. 3 – The time window of w007B on cerebral I/R in rats. Vehicle or agents were administered at different time after reperfusion (IV). (A) Representation of TTC-strained brain slices at 24 h after reperfusion. (B) Infarct volume. (C) Neurological deficit scores. (D) Brain water content. Data were expressed as mean7SEM, n ¼ 10. 44 Po0.01 versus sham; nPo0.05, nnPo0.01 versus I/R. Table 1 – Signs caused by high dose of w007B and honokiol administrated (IP). Animal group
Agenta
Male
w007B NS Honokiol
Female
0 0 60
Solution A
0
w007B NS
0 0
Honokiol Solution A a
Mortality
60 0
Survival times (d)
Symptoms
– – 2 3 5 –
None None Motionless Motionless Motionless None
– – 3 5 6 –
None None Motionless Motionless Motionless None
The dose of both w007B and honokiol were 50 mg/kg.
w007B (50 μg/kg) reduced the infarct volume by 74.6%, which is similar to the results obtained with honokiol in the same mole dose, and the three positive agents (edaravone, 3 mg/kg; butylphthalide, 50 mg/kg; and nimodipine, 1 mg/kg) which reduced the infarct volume by 62.8%, 60.6% and 61.2% respectively. Besides, the effect on neurological deficit scores was similar to that on infarct volume (Fig. 2), suggesting that w007B
has more potent neuroprotective effect than these positive agents. In order to detect the therapeutic time window, w007B was administered at 0, 1, 3 and 5 h after reperfusion. The results showed that it significantly reduced infarct volume when given at 0–3 h after reperfusion (2–5 h after ischemia) (Fig. 3), suggesting that the therapeutic time window is at least 3 h after reperfusion or 5 h after ischemia.
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In order to evaluate the safety of w007B, mice were treated with a single dose w007B (1000 times the highest effective dose). No sign of toxicity or mortality was observed, in
Fig. 4 – The influence on average weight of mice in preliminary acute toxicity experiment. Data were expressed as mean7SEM, n ¼5.
Fig. 5 – The influence on caspase-3 activity in rat brain after cerebral I/R. Casepase-3 activity was expressed as the fold of sham group. Data were expressed as mean7SEM, n¼ 5. 44 Po0.01 versus sham; nnPo0.01 versus vehicle.
contrast with honokiol which, used in the same method, resulted in the death of 60% of the mice within 6 d. These findings strongly suggest that w007B may be safer than honokiol. Apoptosis has been implicated in cerebral ischemic injury (Charriaut-Marlangue et al., 1998) and honokiol has been proved to inhibit endothelial cell apoptosis (Zhang et al., 2007). As a honokiol derivative, w007B may influence the process of apoptosis. Some studies have shown that caspase3 acts as an effector caspase and determines the incidence of apoptosis (Du et al., 1997). In order to investigate the effect of w007B on apoptosis induced by I/R injury, the activity of caspase-3 was monitored. Results showed that w007B reduces caspase-3 activity in I/R-injured hemisphere by 67.2% (Fig. 5). This observation suggests that w007B may exert its neuroprotective effect through the inhibition of neuronal cells apoptosis. NO is an important cytotoxic factor in the process of inflammation (Liu et al., 2002). Our previous studies showed that honokiol inhibited NO production in gial cells induced by LPS (Zhang et al., 2013) and in cortical neurons induced by glutamate and glycine (Hu et al., 2013) and it was therefore supposed that w007B, as a honokiol derivative, may have a similar action mechanism. w007B was indeed found to markedly reduce NO level in I/R tissue (Fig. 6A), which suggests inhibition of NO production during I/R. In addition, NF-κB plays a key role in the initiation of inflammation (Hyun-Ja et al., 2010) and is involved in I/R injury (Khan et al., 2012). It has been demonstrated that NF-κB is activated after transient MCAO, as revealed by the nuclear translocation of the NF-κB subunit p65 (Chen et al., 2009). We observed that w007B inhibited the level of the p65 subunit of NF-κB in the nucleus by 82.0% (Fig. 6B). These data suggested the inhibition of w007B on inflammatory response may be one of crucial mechanisms against I/R injury. Autophagy has been demonstrated to be involved in cerebral injury induced by ischemic stroke (Adhami et al., 2006). Beclin-1 and LC3 have been reported as two reliable markers in the study of autophagy (Rami et al., 2008; Mizushima et al., 2010). LC3 is expressed as three isoforms in mammalian cells, LC3A, LC3B and LC3C, but only LC3B-II
Fig. 6 – The influence on inflammation in cerebral I/R. (A) Effect of w007B on NO release in cerebral hemisphere 24 h after reperfusion. (B) Western blot analysis for the expression of the p65 subunit of NF-κB in cerebral hemisphere and statistical analysis. Data were expressed as mean7SEM, n ¼5, 44 Po0.01 versus sham; nnPo0.01 versus vehicle.
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Fig. 7 – The influence on autophagy in rat brain after cerebral I/R. Western blot analysis for the expression of proteins associate with autophage in cerebral hemisphere 24 h after reperfusion (A). Statistical analysis of Beclin-1, LC3B-II and p62 expression (B, C and D). Data were expressed as mean7SEM, n ¼ 5. 44 Po0.01 versus sham; nPo0.05 versus vehicle.
correlates with increased levels of autophagic vesicles (Barth et al., 2010). In addition, the autophagy-related protein p62/ SQSTM1 is selectively incorporated into autophagosomes through direct binding to LC3, then degraded by autophagy, namely, the p62 protein is unable to be cyclically used (Komatsu et al., 2007). Monitoring of the levels of Beclin-1, LC3B-II and p62 revealed a decrease of both Beclin-1 and LC3B-II, and an increase of p62 levels in the w007B-treated tissue, compared with vehicle-treated tissue. These observations suggest that w007B may exert its inhibitory effect by inactivating autophagy through decreasing Beclin-1 and LC3B-II, and increasing the level of p62, and thus achieve a cerebral protective effect. In conclusion, this study provides some evidence that w007B, a newly synthesized derivative of honokiol, significantly protected rats against ischemia–reperfusion induced brain injury. Besides, w007B has a therapeutic time window of at least 3 h after reperfusion or 5 h after ischemia. Moreover, it is less toxic than its precursor honokiol. It may exert neuroprotective effect, at least partly, through inhibiting the processes of inflammation, apoptosis and autophagy in I/Rinjured brain. All data suggest that w007B can be further developed as a clinical neuroprotective candidate in ischemic stroke.
4.
Experimental procedures
4.1.
Chemicals and agents
w007B and honokiol were provided by Beijing Honghui New Medical Technology Co. Ltd. (China), edaravone injection (Edar) was obtained from Simcere (Nanjing, China), butylphthalide soft capsules (NBP) was obtained from ShiJiaZhuang Pharma Group NBP Pharmaceutical Co. Ltd. (China), nimodipine injection (Nim) was from Bayer Schering Pharma AG (Germany). Honokiol was dissolved in solution A which was composed of polyethylene glycol 400/ethanol/water [40%/10%/50% (v/v/v)].
4.2.
Animals
ICR mice (19–21 g) and male SD rats (240–270 g) were provided by Department of Laboratory Animal Science, Peking University Health Science Center. All experimental procedures were approved by the Beijing Committee on Animal Care and Use. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Peking University Health Science Center (Permit number: LA2013-69). All animals were kept on a 12-h light/12-h dark regime, with free access to food and water.
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4.3.
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MCAO reperfusion model in rats
Transient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) as described previously (Hu et al., 2013) under anesthesia with chloral hydrate (350 mg/kg, IP), the filament was removed 2 h after MCAO. Sham-operated control rats received the same procedure except filament induction. The body temperature of rats was maintained and monitored at 3770.5 1C by holding operation table, and relative regional Cerebral Blood Flow (rCBF) was monitored with laser Doppler measurement (PeriFlux System 5000) during the surgery. A minimum reduction of 70% of the initial reading of LDF was considered as a successful occlusion of MCA perfusion territory (DiNapoli et al., 2006). Experiment 1: Ninety rats were randomly assigned to nine groups: sham group, vehicle group (normal saline), w007Btreated groups in different doses (2, 10, 50 mg/kg), honokioltreated group (28.5 mg/kg), edaravone-treated group (3 mg/kg), NBP-treated group (50 mg/kg) and nimodipine-treated group (1 mg/kg), 10 rats per group. Vehicle or agents were administered immediately after reperfusion intravenously. Experiment 2: Sixty rats were randomly assigned to six groups: sham group, vehicle group (normal saline), w007Btreated groups at different time points (0, 1, 3, 5 h after reperfusion), 10 rats per group. Animals were treated at different times after reperfusion intravenously. Neurological deficit was evaluated 24 h after reperfusion according to the Longa's method (Longa et al., 1989). Then the brains were dissected and cut into 5 coronal slices, 2-mm each, the slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC, Amresco, USA) for 15 min at 37 1C, according to the previous method (Bederson et al., 1986) with slight modification, followed by immersion-fixation in 4% paraformaldehyde. TTC-stained sections were photographed and the digital images were analyzed using image analysis software (NIH ImageJ software). Infarct areas were first measured using image analysis software and then compiled to obtain the infarct volume (mm3) per brain. The lesion volumes were calculated as a percentage of the contralateral hemisphere volume to compensate for the effect of brain edema using the following formula (Tatlisumak et al., 1998): f½total infarct volume ðthe volume of intact ipsilateral hemisphere the volume of intact contralateral hemisphereÞ =contralateral hemisphere volumeg 100%:
Cerebral edema extent was determined by measuring the brain water content according to the wet–dry method as described in previous reports (Chao et al., 2010) with some modifications. In brief, brains were immediately weighed to obtain wet weight after removal, then dried in an oven at 110 1C for 16 h and weighed again to obtain the dry weight. Brain water content was calculated as follows: Brain water content ð%Þ ¼ ½ðwet weightdry weightÞ=wet weight 100%:
4.4.
Preliminary acute toxicity
Forty ICR mice were randomly assigned to four groups: w007B-treated group, vehicle control group (normal saline), honokiol-treated group, and vehicle control group of honokiol (solution A). Following a single dose of the agents (50 mg/kg; IP), mice were observed for 14 d. All the animals were weighed before the treatment and the following days. Mortality, survival times (days) and symptoms induced were recorded within 14 d.
4.5.
Determination of casepase-3 activity
The influence of w007B on I/R-induced apoptosis was evaluated by detecting the activity of caspase-3 using a Colorimetric Assay Kit (KeyGen Biotech, Nanjing, China). Proteins were extracted from fresh ischemia ipsilateral cortical samples 24 h after reperfusion. Tissues were homogenized in cold lysis buffer containing 25 mM HEPES, pH 7.5, 5 mM MgCl2, 2 mM EDTA, 0.1% Triton 100, 2 mM dithiothreitol and 1 mM PMSF. The homogenates were centrifuged at 12,000g for 30 min at 4 1C, and the supernatants were collected. The protein concentration was determined by BCA assay (M&C Gene Technology, Beijing, China) and adjusted to 10 mg/ml. Then 500 mg proteins were added to 50 ml 2 reaction buffer supplement with 5 ml caspase-3 substrate and incubated at 37 1C away from light for 4 h. Caspases-3 activities were quantified spectrophotometrically at a wavelength of 405 nm. The activity of caspase-3 was expressed as the fold of the activity of sham group.
4.6.
Determination of inflammatory markers
The influence of w007B on I/R-induced inflammatory molecule of NO was determined by 2,3-diaminonaphthalene (DAN; Fluka) as previously reported (Misko et al., 1993). In brief, cerebrum was collected and homogenized in cold PBS; the supernatant was obtained by centrifugation at 1000g for 10 min. All procedures were performed at 4 1C. Supernatants were either used directly or stored at 80 1C. Protein concentrations were determined by the BCA method, and 100 ml of the supernatants of brains tissue supernatants was transferred to 96-well plates, mixed immediately with the solution of DAN (20 μl; 50 μg/ml in 0.62 M HCl), and incubated for 15 min in the dark at 25 1C. The fluorescence was measured with a FlexStation3 microplate reader with excitation at 365 nm and emission at 450 nm after adding NaOH (100 ml; 0.28 M). The nitrite concentration was determined based on the standard regression line for NaNO2. The nuclear translocation level of p65 subunit of NF-κB was detected through western blotting, nucleus proteins extracted with cold Nuclear and Cytoplasmic Extraction Kit (CWBIO, Beijing, China) were analyzed by Western blot. Western blot procedure followed standard protocols (Shimamura et al., 2006). Shortly, 35 μg of total protein were separated in 12% SDSpolyacrylamide gel electrophoresis (PAGE) and blotted onto PVDF membranes (Millipore, Billerica, MA, USA). Antibodies included Mouse anti-NF-κB p65 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Histone H1 (1:500, Zhongshan Golden Bridge Bio-technology, Beijing, China) and appropriate secondary antibodies (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein bands were detected with
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a chemiluminescent reagent (Millipore, Billerica, MA, USA). Optical density of the bands was quantified in Image Lab software, and the housekeeping protein Histone H1 was used as a control and tested simultaneously by mouse monoclonal anti-Histone H1 antibody.
4.7.
Determination of autophagy-related proteins
The effect of w007B on I/R-induced autophagy by western blot analysis with autophagy specific antibodies against Beclin-1, LC3B-II and p62, proteins extracted from the brains in cold RIPA Lysis Buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% C24H40O4 Na, 0.1% SDS, 2 mM Na4P2O7, 25 mM β-glycerophosphate, 1 mM Na3VO4 and 1 mM PMSF) were subjected to Western blot analysis. Western blotting procedure followed standard protocols (Shimamura et al., 2006): 35 μg of total protein were separated in 15% or 12% SDS-PAGE and blotted onto PVDF membranes (Millipore, Billerica, MA, USA). Antibodies included rabbit anti-Beclin-1 antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-LC3B antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-p62 antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-β-actin (1:1000, Zhongshan Golden Bridge Bio-technology, Beijing, China), and appropriate secondary antibodies (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein bands were detected with a chemiluminescent reagent (Millipore, Billerica, MA, USA). Optical density of the bands was quantified in Image Lab software, and the housekeeping protein β-actin was used as a control and tested simultaneously by mouse monoclonal anti-β-actin antibody.
4.8.
Statistical analysis
All data were expressed as mean7SEM. The one way ANOVA was used to assess differences among groups, Significant differences between groups at each time point were assessed by Student's t-test. Po0.05 was considered to be statistically significant.
Acknowledgments This work was supported by the Chinese National Technology Graveness Special Purpose Fund (2009zx09102-146) and the National Natural Science Foundation of China (61071002).
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