Just Accepted by International Journal of Neuroscience
Remote limb ischemic postconditioning protects mouse brain against cerebral ischemia/reperfusion injury via upregulating expression of Nrf2, HO-1, NQO-1 in mice Pan Li, Likai Su, Xiaofang Li, Weiying Di, Xiangjian Zhang, Cong Zhang, Tingting He, Xingyuan Zhu, Ye Zhang, Yaoru Li doi:10.3109/00207454.2015.1042973
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Abstract Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention, which has been discovered to reduce ischemia/reperfusion (I/R) injury in heart, kidney, brain and skeletal muscle experimentally. However, its potential protective mechanisms have not been well elucidated. The aim of this study was to investigate the protective effect of RIPostC in cerebral I/R injury and explore the new putative mechanisms of neuroprotection elicited by it. Focal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in male CD1 mice. RIPostC was generated by three cycles of 5 minute reperfusion/5 minute occlusion of the bilateral femoral artery on the bilateral limbs at the onset of middle cerebral artery reperfusion. RIPostC significantly improved neurological outcome, lessened infarct volume and brain edema, up-regulated the expression of Nrf2, HO-1, NQO-1 and activity of SOD, down-regulaed the formation of MDA ( P < 0.05). Taken together, these findings demonstrated that RIPostC protected the brain from I/R injury after focal cerebral ischemia by reducing oxidative stress and activating of the Nrf2-ARE pathway.
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Publisher: Taylor & Francis Journal: International Journal of Neuroscience DOI: http://dx.doi.org/10.3109/00207454.2015.1042973
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Dear Editor:
It is our great honor to submit the results of our recently completed
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against cerebral ischemia/reperfusion injury via upregulating expression of Nrf2, HO-1, NQO-1 in mice” to International Journal of Neuroscience: Accumulating evidence indicated that oxidative stress plays a key role in pathophysiological mechanism of cerebral ischemia-reperfusion (I/R) injury.
As
many
studies
has
been
proved,
remote
ischemic
postconditioning (RIPostC) could reduce ischemia/reperfusion (I/R)
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research “Remote limb ischemic postconditioning protects mouse brain
injury, and the generation of ROS by protective stimuli in the limb may as an important trigger involved in the neuroprotection of RIPostC. This study investigated the protective effect of RIPostC in cerebral I/R injury and explore the new putative mechanisms of neuroprotection elicited by it. Our results showed RIPostC significantly improved neurological outcome, lessened infarct volume and brain edema, up-regulated the expression of Nrf2, HO-1, NQO-1 and activities of SOD, down-regulaed the formation of MDA. All authors have seen the manuscript and approved to submit to your journal. This result has not been published and is not being considered for publication elsewhere in whole or in part in any language. 1
All persons mentioned in the acknowledgment have seen and approved mention of their names in the article. This manuscript is in accordance with the Authorship statement of ethical standards for manuscripts
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submitted to International Journal of Neuroscience. The authors declare no conflict of interest. Thank you very much for your attention and
Likai Su
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Sincerely yours
Remote limb ischemic postconditioning protects mouse brain against cerebral ischemia/reperfusion injury via upregulating expression of
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consideration.
Nrf2, HO-1, NQO-1 in mice
Pan Li,1 Likai Su,1 Xiaofang Li,1 Weiying Di,1 Xiangjian Zhang,2,3,4 Cong
Zhang,2 Tingting He,2 Xingyuan Zhu,2 Ye Zhang,2 Yaoru Li2
1
Department of Neurology, Affiliated Hospital of Hebei University;
Baoding, Hebei 071000, PR China 2
Department of Neurology, Second Hospital of Hebei Medical University;
Shijiazhuang, Hebei 050000, PR China 3
Hebei Collaborative Innovation Center for Cardio-cerebrovascular
Disease, Shijiazhuang, Hebei 050000, PR China 4
Hebei Key Laboratory for Neurology; Shijiazhuang, Hebei 050000, PR 2
China ﹡
Corresponding author: Likai Su. Department of Neurology, Affiliated
Hospital of Hebei University, 212 Yuhua East Road, Baoding, Hebei China.
E-mail
addresses:
Abstract
Tel:
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Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention, which has been discovered to reduce
ischemia/reperfusion (I/R) injury in heart, kidney, brain and skeletal muscle experimentally. However, its potential protective mechanisms have not been well elucidated. The aim of this study was to investigate
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8613832203238. Fax: + 8603125981036.
[email protected].
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071000,
the protective effect of RIPostC in cerebral I/R injury and explore the new putative mechanisms of neuroprotection elicited by it. Focal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in male CD1 mice. RIPostC was generated by three cycles of 5 minute reperfusion/5 minute occlusion of the bilateral femoral artery on the bilateral limbs at the onset of middle cerebral artery reperfusion. RIPostC significantly improved neurological outcome, lessened infarct volume and brain edema, up-regulated the expression of Nrf2, HO-1, NQO-1 and activity of SOD, down-regulaed the formation of MDA ( P < 0.05). Taken together, these findings demonstrated that RIPostC protected 3
the brain from I/R injury after focal cerebral ischemia by reducing oxidative stress and activating of the Nrf2-ARE pathway.
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Keywords: Cerebral ischemia, Ischemia/reperfusion injury; Remote
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ischemic postconditioning; Neuroprotection; Anti-oxidative stress
4
Introduction Ischemic conditioning is a form of endogenous protection which
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targets ischemia/reperfusion (I/R) injury induced by transient, sublethal ischemia in a tissue. Organs with high sensitivity to ischemia, such as the
targets for potential therapeutic applications of ischemic conditioning.
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Ischemic pre-/post-conditioning refers to the application of brief
subcritical ischemia in brain before (preconditioning) or after (postconditioning) a prolonged destructive ischemic insult, capable of triggerating innate mechanisms to protect against brain injury after stroke [1-4]. Although numerous preclinical investigations have studied the
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heart and the brain, represent the most potentially and vitally promising
potential benefits of pre-/post-conditioning with promising results, a less invasive methods was still required for potential clinical application. One of the most remarkable breakthroughs in the ischemic conditioning field is pre-/post-conditioning can be performed at a distance or remote from the ischemic target organ because it has the most clinical relevance and translational potential property [5,6]. Remote ischemic postconditioning (RIPostC), which is induced by cycles of a few minutes of I/R applied to a distal artery territory either immediately after the beginning of brain reperfusion or just a few minutes before reperfusion, is an emerging concept for brain I/R injury treatment. However, its potential protective 5
mechanisms have not been well established [7]. Ischemic stroke is a destructive cerebrovascular disease and a leading cause of death and disability worldwide [8]. Although several
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compounds with neuroprotective qualities have been identified in experimental settings, their transition to clinical practice is unsuccessful
RIPostC functions is still urgently needed to be further studied.
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Accumulating evidence indicated that oxidative stress plays a key role in
pathophysiological mechanism of cerebral I/R injury [10,11]. Excessive accumulation of toxic free radicals such as reactive oxygen species (ROS) could trigger diverse signaling pathways and lead to oxidative damage, contributing to the deterioration and progression of ischemic injury after
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[9]. Due to its translational potential property, the mechanism by which
stroke [12]. Superoxide dismutase (SOD), an endogenous anti-oxidant enzyme, plays a crucial role in the oxidative and antioxidative balance to protect organisms from free radical attack. Recent reports showed that ischemic postconditioning attenuates the production of ROS, inhibits malondialdehyde (MDA) increase level [13] and enhances the activity of SOD [14].
Nuclear factor erythroid 2-related factor 2 (Nrf2) is considered to be
a multi-organ protector and is widely deemed to play a vital role in regulating its endogenous antioxidant capacity in cerebral ischemia [15,16]. The antioxidant response element (ARE) is a cis-acting 6
regulatory element that governs the expression of anti-oxidant enzymes and phase II detoxification enzymes. Nrf2 can coordinately regulate the ARE containing genes expression. Differ with other enzymes, heme
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oxygenase 1(HO-1) and quinone oxidoreductase-1(NQO-1) expression is reduced significantly in Nrf2 knockdown mice and is abolished by Nrf2
preconditioning may produce cardioprotection through an enhanced
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HO-1 expression partly via Nrf2 translocation [19].
This study was designed to investigate whether RIPostC, performed by three cycles of occlusion and release of the femoral artery, ameliorated oxidative stress damage in the mouse model of transient middle cerebral artery occlusion (tMCAO), and whether the therapeutic benefit of it was
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disruption [17,18]. It has been reported that delayed remote ischemic
associated with the activation of the Nrf2-ARE pathway. Materials and methods
Experimental animals and grouping Adult male CD1 mice (25 - 30 g) were purchased from the Vital
River Laboratory Animal Technology Co. Ltd, Beijing, China. The experimental procedures were approved by the institutional animal care and use committee and the local experimental ethics committee and conformed to internationally accept ethical standards. All mice were fed a standard diet and water, treated humanely, and maintained at 22±3°C with 12/12 h light/dark cycle, and they were allowed to acclimatize the 7
new surrounding for at least 3 days ahead of any experimentation. Mice were divided into three groups randomly. Sham group: the sham-operated group, in which mice underwent the same surgical
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procedure without inserting a filament and inducing in bilateral hind limbs by occluding and releasing the femoral arteries; the tMCAO group:
group: in which mice were subjected to focal cerebral ischemia and limb
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ischemic postconditioning.
Transient focal cerebral ischemia and remote limb ischemic postconditioning
Transient middle cerebral artery occlusion (tMCAO) surgical procedures were performed as described previously [20]. The tMCAO
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in which mice were subjected to focal cerebral ischemia; the RIPostC
model was induced by right middle cerebral artery occlusion (MCAO) for 1 h. An incision along the vascular route was conducted on femoral triangle of the hind limb, and the bilateral femoral arteries were exposed before MCA occlusion operation. In the RIPostC group, reversible hind limb ischemia was induced in bilateral hind limbs by occluding and releasing the femoral artery for three cycles (occlusion/release 5 min/5 min) immediately after stroke onset. Neurobehavioral evaluation A neurological test was evaluated by an investigator blinded to the experimental groups at 24 h after operation following a modified point 8
scale system based on a 28 point scale of focal neurological scores (FNS) [21], which was based on the following tests: 1, body symmetry, 0 to 4 points; 2, gait, 0 to 4 points; 3, climbing, 0 to 4 points; 4, circling
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behavior, 0 to 4 points; 5, frontal limb symmetry, 0 to 4 points; 6, compulsory circling, 0 to 4 points; 7, whisker response, 0 to 4 points. The
higher score, the more severe impairment of motion injury (the total score
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for each animal is 0 to 28). (n = 15 per group). Measurement of Brain Water Content
Brain water content was measured by the standard wet-dry method at 24 h after operation [22], as described previously [20]. (n = 6 per group).
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7 individual test scores were summed up at the end of the evaluation. The
Infarct volume assessment
Infarct volume was measured at 24 h after reperfusion which was
determined by 2,3,5-triphenyltetrazolium chloride (TTC) [23], as described previously [23]. To offset the effect of brain edema, the corrected infarct volume was calculated as follows: percentage of corrected infarct volume = {[total infarct volume ipsilateral hemisphere
−
−
(the volume of intact
the volume of intact contralateral hemisphere)] /
contralateral hemisphere volume} × 100%. ( 5 slices/mice n = 6 per group). Immunohistochemistry (IHC) 9
Standard Immunohistochemistry procedure was performed as previously described [20] with a modification of antibodies: After blocked in 3% H2O2 and 3% normal goat serum, the brain sections were
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incubated with Nrf2 rabbit polyclonal antibody (1:50, Santa Cruz Biotechnology), HO-1 rabbit polyclonal antibody (1:100, Enzo Life
Technology), in 0.01 mol/L phosphate-buffered saline overnight. Five
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visual fields of ischemic region of the infarct were selected and the immunoreactive cells were counted under a 400 × light microscope. Counting of the positive cells was performed by an investigator blinded to the experimental groups. (n = 3 for each group)
Reverse Transcription-Polymerase Chain Reaction (RT-qPCR)
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Sciences, Inc), and NQO-1 rabbit polyclonal antibody (1:80, Bioworld
Mice (n=6 in each group) were anesthetized and the ischemic cortex
in ipsilateral hemisphere was harvested and frozen in liquid nitrogen at 24 h after reperfusion. As described previously [20], the mRNA expression of Nrf2, HO-1 and NQO-1 were detected by qRT-PCR (MX 3005P, USA) in the presence of a fluorescent dye (SYBR Green I; Cwbio). Forward and reverse primers were 5’-GACAAACATTCAAGCCGATTAGAGG-3’ and
3’-ACTTTATTCTTCCCTCTCCTGCGT-5’ for Nrf2, 5’-TACTCATCCTGAGCTGCTGGT-3’ 3’-TGGACGAAGAAACTCTGTCTGT 10
and for
HO-1,
5’-GGTATTACGATCCTCCCTCAACATC-3’ GAGTACCTCCCATCCTCTCTTCTTC-3’
and for
5’NQO-1,
5’-TGAACGGGAAGCTCACTGG-3’, and
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5’-GCTTCACCACCTTCTTGATGTC-3 for GAPDH. (n = 6 per group) Western Blotting
reperfusion and the ischemic hemisphere of cortex was harvested (from
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an area between 3 and 6 mm frontal to the posterior pole). For the nuclear
protein of Nrf2, the tissue was homogenized in an ice-cold lysis buffer A [10mmol/L
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
[(HEPES), pH 7.8, 10 mmol/L KCl, 2 mmol/L MgCl2, 0.1 mmol/L ethylene diamine tetraacetic acid (EDTA), 1 mmol/L dithiothreitol (DTT),
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All the mice (n=4 in each group) were killed 24 hours after
and 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF)] for 20 min. 10 % Nonidet P-40 (NP-40) was added to the homogenates, and the mixture was centrifuged for 3 min at 10,000 rpm at 4 °C. The supernatant was harvest as a cytosolic fraction for nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, 4-hydroxynonenal (4-HNE) and alpha (IκBα) assays. The pellets were homogenized in ice-cold lysis buffer C (20 mmol/L HEPES, pH 7.8, 300 mmol/L NaCL, 1 mmol/L EDTA, 0.1 mmol/L EGTA, and 0.1 mmol/L PMSF twice) for 40 min. Then centrifuged for 15 min at 14,000 rpm at 4 °C. The supernatant was collected and added PMSF to the final concentration 1 mmol/L as the 11
nuclear protein of Nrf2 (Nrf2,1:100, Santa Cruz), and polyclonal rabbit Lamin B1 antibody (1:1000, Santa Cruz) was used as an internal control. The extracting and analysis of the total protein of HO-1 and NQO-1
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were described as previous reports [20]. Antibodies used here were insteaded by rabbit polyclonal antibody anti-HO-1 (1:500, 1:800, ENZO
(1:10000, Bioworld Technology) (n = 4 in each group) .
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Determination of SOD and MDA levels
Cortical tissues from six mice of each group were collected at 24 hours after operation. The samples were rinsed, weighed, and then homogenized in 9 volumes of 9 g/L ice-cold saline for 10 min using a Dounce Tissue Grinder (Kimble and Kontes, Vineland, NJ, USA).
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antibody), anti-NQO-1, (1:200 Bioworld Technology), and anti-GAPDH
Supernatant homogenate was collected after centrifugation at 4000 rpm/min for 10 min at 4 °C. SOD and MDA levels were measured using an assay kits (A001 and A003; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturers’ instructions. (n = 6 in each group)
Statistical Analysis
All data were analyzed by SPSS 13.0 software, while all quantitative
data were represented as mean ± SD. Statistical comparisons were performed by one-way ANOVA followed by Student-Newman-Keuls test (SNK) for intergroup comparisons. For neurologic deficit scores, 12
Mann-Whitney U test was used for comparisons between groups. P < 0.05 was considered as statistically significant. Results
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RIPostC improved the neurological deficit The Average of neurological score of sham-operated mice was zero.
highest compared with the Sham group (P < 0.05). Compared with
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tMCAO group, there was a significant improvement in neurological function scores in the RIPostC group (P < 0.05) (Figure. 1A). RIPostC reduced the brain edema
Brain water content at 24 hours after operation was shown in Fig. 1B. Ipsilateral Brain water content of Sham group was 78.46% ± 0.77%.
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Whereas the neurological deficit score in the tMCAO group was the
Following ischemia, it shown higher in the tMCAO group ( P < 0.05 vs. Sham). Following RIPostC treatment, as compared with tMCAO group, there was a significant reduction in brain water content in the RIPostC group (81.68% ± 0.66% vs. 84.23 ± 0.63%，P < 0.05). RIPostC reduced infarct volume No infarction was observed in Sham group. Extensive lesion was
found in both striatum and cortex in tMCAO group. Following ischemia, the infarct volume got higher in the tMCAO group ( P < 0.05 vs. Sham). Fig. 1C showing typical photographs of 2% TTC-stained sections from tMCAO and RIPostC-treated mice. The infarct volume was significantly 13
reduced from 42.84% ± 3.98% in tMCAO group to 25.89% ± 3.63% in the RIPostC group ( P < 0.05, Fig. 1D). RIPostC increased the expression of Nrf2, HO-1 and NQO-1 after
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cerebral I/R To investigated the endogenous expression of Nrf2, HO-1 and
western blot and RT-qPCR were performed, respectively. The number of
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positive cells was counted manually and intergroup comparisons were performed. The immunohistochemical staining for Nrf2, HO-1 and NQO-1 of each group was done at 24 h after operation and was shown in Fig. 2A. Few cells were expressing Nrf2, HO-1 and NQO-1 in the cortex of Sham group indicating a low expression in the nonischemic cortex. In
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NQO-1 in ischemic hemisphere of each group, immunohistochemistry,
contrast, the number of positive cells in the ischemic cortex of tMCAO mice was significantly increased compared with Sham group ( P < 0.05). In RIPostC groups, the number of positive cells of Nrf2, HO-1 and NQO-1 was significantly increased than the tMCAO group ( P < 0.05) (Figure. 2B). Results from Western blot analysis and RT-qPCR analysis showed the same patterns at protein levels and the mRNA level of Nrf2, HO-1
and
NQO-1
were
in
according
with
the
results
of
immunohistochemistry ( P < 0.05, Fig. 3A-D). RIPostC increased the activity of SOD and decreased MDA content The activities of SOD were up-regulated and the production of 14
MDA were increased at 24 h after operation in the tMCAO group compared with Sham group ( P < 0.05). Compared with the tMCAO group, we also found that RIPostC can significantly increased the
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activities of SOD and decreased the production of MDA ( P < 0.05). (Figure. 4A,B)
Stroke is a life-threatening disease causing high mortality and
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disability worldwide. Many studies had been proved that RIPostC could
reduce ischemia/reperfusion (I/R) injury and the generation of ROS by protective stimuli in the limb which may be considered as an important trigger involving in the neuroprotection of RIPostC [13]. Our study showed that RIPostC was an effective therapeutic intervention against
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Discussion
cerebral I/R injury in a mouse tMCAO model at 24 h after operation as evidenced by improving neurological outcome, lessening infarct volume and brain edema and exerting anti-oxidant effects. Furthermore, the anti-oxidant effects may be through activating of the Nrf2-ARE pathway. When the oxidative stress occurred, the rapid overproduction of free
radicals overwhelms the detoxification and scavenging capacity of cellular anti-oxidative enzymes like SOD, issuing in a severe damage to neurons [24]. SOD is one of the key free radical scavengers, which detoxifies superoxide radicals to hydrogen peroxide specifically to protect organisms from free radical attack in the body. MDA is one of oxidative 15
stress markers, which expression level reflects the rate and extent of lipid peroxidation as well as the capacity for eliminating free radicals [25]. Compared with the tMCAO group, we also found that RIPostC can
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significantly increase the activities of SOD and decrease the production of MDA in the present study, indicating that the neuroprotection
The overexpression of Nrf2 has become a potential therapeutic
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avenue for ischemic stroke. The antioxidant response element (ARE) is a
cis-acting regulatory element that controlls the expression of phase II detoxification
enzymes
[e.g.
heme
oxygenase-1
(HO-1),
NAD(P)H:quinone oxidoreductase-1 (NQO-1), superoxide dismutase 1 (SOD1), glutathioneS-transferases (GSTs), etc][26]. Nrf2, belonging to
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conferred by RIPostC is due to its anti-oxidative effect.
Cap‘n’collar/basic-leucine zipper family, regulates the ARE containing genes. Numerous antioxidants have been shown to protect the brain from the injury caused by brain ischemia and reperfusion throgh the Nrf2/ARE pathway [27,28]. The chaperone protein HO-1, in concert with cytochrome p450, oxidatively cleaves heme to biliverdin that is subsequently converted to bilirubin. As we all known, both biliverdin and bilirubin have shown anti-oxidative and free radical scavenging properties [29,30]. It is proved that increasing the activity of HO-1 exerts neuroprotective against oxidative and excitotoxic insults relevant to stroke both in vivo and in vitro study [27,31]. NQO-1 is generally 16
considered a detoxifying enzyme because its ability to decrease reactive quinones and quinone imines to less toxic hydroquinonesand less reactive [32]. Various kinds of small molecules like tertiary butyl hydroquinone
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(tBHQ) and sulforaphane, antioxidants are known to activate NQO-1 mediated via ARE [33]. NQO-1 is highly expressed in the lung
antioxidive protection [34]. Our study showed that the expression of Nrf2,
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HO-1 and NQO-1 in ischemic cortex at 24 h after reperfusion were
increased compared with Sham group, and RIPostC significantly increased the expression of Nrf2, HO-1 and NQO-1 compared with tMCAO group, suggesting that RIPostC might have a potent anti-oxidative effect on cerebral I/R through the activation of the
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epithelium and tissues as well as central nervous system that require high
Nrf2-ARE pathway.
It is evident from reports showing that RIPostC is a new
experimented non-pharmacologic intervention and is a potent therapeutic strategy that can be easily and quickly administered to protect the brain
from ischemic damage. However, RIPostC and its fundamental biology are not well understood, and studies to date are largely observational [32,33]. There are plenty of mechanistic work remains to be done not only to provide a theoretical basis for its clinical application, but also to illuminate further pathways involved in the neuroprotection induced by limb ischemic postconditioning. By the identification of those pathways 17
exerted by RIPostC, it will be possible to find out new druggable targets for developing effective strategies to treat ischemic stroke. Our study demonstrated that RIPostC is a new clinically translatable, feasible, and
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safe approach to treat cerebral ischemia, and provided a theoretical basis for its clinical application.
in cerebral I/R due to its anti-oxidative effect and the anti-oxidative effect
Conflict of interest
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may be through activating the Nrf2-ARE pathway.
The authors declared that there are no conflicts of interest.
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In conclusion, our results showed that RIPostC has beneficial effects
Acknowledgments
This work was funded by the Medical Disciplines Funded Projects of Hebei University, China (Grant no. 11ZF006).
18
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13. Sun HY, Wang NP, Kerendi F, et al. Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload. American Journal of Physiology Heart and Circulatory Physiology 2005;288(4):H1900-1908. 20
14. Li ZY, Liu B, Yu J, et al. Ischaemic postconditioning rescues brain injury caused by focal ischaemia/reperfusion via attenuation of protein oxidization. J Int Med Res 2012;40(3):954-66.
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18. Innamorato NG, Rojo AI, García-Yagüe AJ, et al. The transcription
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20. Wang L, Li Z, Zhang X, et al. Protective effect of shikonin in experimental ischemic stroke: attenuated TLR4, p-p38MAPK, NF-kB, TNF-αand MMP-9 expression, up-regulated claudin-5 expression, 21
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Free Radical Biology and Medicine 2013;65:1012-1022. 28. Wu J, Li Q, Wang X, et al. Neuroprotection by Curcumin in Ischemic Brain
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disease states. Acta. Biochim Pol 2005;52,273-284.
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Figure legends: Fig. 1. Effect of RIPostC administration on ischemic mice brains. (A) RIPostC improved neurological deficits (n = 15). The neurological deficit
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scores were significantly higher neurological deficit scores in tMCAO group compared with the Sham group and lower in RIPostC group
tMCAO). (B) The water content of ipsilateral hemispheres was higher in
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tMCAO group compared with Sham group and reduced in RIPostC group
compared with tMCAO group (n = 6). ( ☆P < 0.05 vs. Sham; ★P < 0.05 vs. tMCAO). (C) Representative images of TTC staining at brain slices at 24 h after reperfusion (n = 6). The pale region was the infarct brain tissue and the red region was normal. (D) Following ischemia, the infarct volume got higher in the tMCAO group (n = 6). ( ☆P < 0.05 vs. Sham).
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compared with the tMCAO group ( ☆P < 0.05 vs. Sham; ★P < 0.05 vs.
The infarct volume was lessened ( ★P < 0.05 vs. tMCAO)
24
Fig.2. Immunohistochemical staining of Nrf2, HO-1 and NQO-1 in the cerebral cortex at 24 h after reperfusion (n = 3). (A) Representative images for immunoreactive staining of Nrf2, HO-1 and NQO-1 in
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different groups. (400×magnification). (B) Quantification of the number of immunoreactive cells in different groups. The number of
increased in tMCAO and RIPostC. For Nrf2, ☆P < 0.05 vs. Sham; ★P
Remote limb ischemic postconditioning protects mouse brain against cerebral ischemia/reperfusion injury via upregulating expression of Nrf2, HO-1, NQO-1 in mice Pan Li, Likai Su, Xiaofang Li, Weiying Di, Xiangjian Zhang, Cong Zhang, Tingting He, Xingyuan Zhu, Ye Zhang, Yaoru Li doi:10.3109/00207454.2015.1042973
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Abstract Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention, which has been discovered to reduce ischemia/reperfusion (I/R) injury in heart, kidney, brain and skeletal muscle experimentally. However, its potential protective mechanisms have not been well elucidated. The aim of this study was to investigate the protective effect of RIPostC in cerebral I/R injury and explore the new putative mechanisms of neuroprotection elicited by it. Focal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in male CD1 mice. RIPostC was generated by three cycles of 5 minute reperfusion/5 minute occlusion of the bilateral femoral artery on the bilateral limbs at the onset of middle cerebral artery reperfusion. RIPostC significantly improved neurological outcome, lessened infarct volume and brain edema, up-regulated the expression of Nrf2, HO-1, NQO-1 and activity of SOD, down-regulaed the formation of MDA ( P < 0.05). Taken together, these findings demonstrated that RIPostC protected the brain from I/R injury after focal cerebral ischemia by reducing oxidative stress and activating of the Nrf2-ARE pathway.
© 2015 Informa Healthcare USA, Inc. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
Publisher: Taylor & Francis Journal: International Journal of Neuroscience DOI: http://dx.doi.org/10.3109/00207454.2015.1042973
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Dear Editor:
It is our great honor to submit the results of our recently completed
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against cerebral ischemia/reperfusion injury via upregulating expression of Nrf2, HO-1, NQO-1 in mice” to International Journal of Neuroscience: Accumulating evidence indicated that oxidative stress plays a key role in pathophysiological mechanism of cerebral ischemia-reperfusion (I/R) injury.
As
many
studies
has
been
proved,
remote
ischemic
postconditioning (RIPostC) could reduce ischemia/reperfusion (I/R)
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research “Remote limb ischemic postconditioning protects mouse brain
injury, and the generation of ROS by protective stimuli in the limb may as an important trigger involved in the neuroprotection of RIPostC. This study investigated the protective effect of RIPostC in cerebral I/R injury and explore the new putative mechanisms of neuroprotection elicited by it. Our results showed RIPostC significantly improved neurological outcome, lessened infarct volume and brain edema, up-regulated the expression of Nrf2, HO-1, NQO-1 and activities of SOD, down-regulaed the formation of MDA. All authors have seen the manuscript and approved to submit to your journal. This result has not been published and is not being considered for publication elsewhere in whole or in part in any language. 1
All persons mentioned in the acknowledgment have seen and approved mention of their names in the article. This manuscript is in accordance with the Authorship statement of ethical standards for manuscripts
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submitted to International Journal of Neuroscience. The authors declare no conflict of interest. Thank you very much for your attention and
Likai Su
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Sincerely yours
Remote limb ischemic postconditioning protects mouse brain against cerebral ischemia/reperfusion injury via upregulating expression of
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consideration.
Nrf2, HO-1, NQO-1 in mice
Pan Li,1 Likai Su,1 Xiaofang Li,1 Weiying Di,1 Xiangjian Zhang,2,3,4 Cong
Zhang,2 Tingting He,2 Xingyuan Zhu,2 Ye Zhang,2 Yaoru Li2
1
Department of Neurology, Affiliated Hospital of Hebei University;
Baoding, Hebei 071000, PR China 2
Department of Neurology, Second Hospital of Hebei Medical University;
Shijiazhuang, Hebei 050000, PR China 3
Hebei Collaborative Innovation Center for Cardio-cerebrovascular
Disease, Shijiazhuang, Hebei 050000, PR China 4
Hebei Key Laboratory for Neurology; Shijiazhuang, Hebei 050000, PR 2
China ﹡
Corresponding author: Likai Su. Department of Neurology, Affiliated
Hospital of Hebei University, 212 Yuhua East Road, Baoding, Hebei China.
addresses:
Abstract
Tel:
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Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention, which has been discovered to reduce
ischemia/reperfusion (I/R) injury in heart, kidney, brain and skeletal muscle experimentally. However, its potential protective mechanisms have not been well elucidated. The aim of this study was to investigate
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8613832203238. Fax: + 8603125981036.
[email protected].
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071000,
the protective effect of RIPostC in cerebral I/R injury and explore the new putative mechanisms of neuroprotection elicited by it. Focal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in male CD1 mice. RIPostC was generated by three cycles of 5 minute reperfusion/5 minute occlusion of the bilateral femoral artery on the bilateral limbs at the onset of middle cerebral artery reperfusion. RIPostC significantly improved neurological outcome, lessened infarct volume and brain edema, up-regulated the expression of Nrf2, HO-1, NQO-1 and activity of SOD, down-regulaed the formation of MDA ( P < 0.05). Taken together, these findings demonstrated that RIPostC protected 3
the brain from I/R injury after focal cerebral ischemia by reducing oxidative stress and activating of the Nrf2-ARE pathway.
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Keywords: Cerebral ischemia, Ischemia/reperfusion injury; Remote
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ischemic postconditioning; Neuroprotection; Anti-oxidative stress
4
Introduction Ischemic conditioning is a form of endogenous protection which
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targets ischemia/reperfusion (I/R) injury induced by transient, sublethal ischemia in a tissue. Organs with high sensitivity to ischemia, such as the
targets for potential therapeutic applications of ischemic conditioning.
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Ischemic pre-/post-conditioning refers to the application of brief
subcritical ischemia in brain before (preconditioning) or after (postconditioning) a prolonged destructive ischemic insult, capable of triggerating innate mechanisms to protect against brain injury after stroke [1-4]. Although numerous preclinical investigations have studied the
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heart and the brain, represent the most potentially and vitally promising
potential benefits of pre-/post-conditioning with promising results, a less invasive methods was still required for potential clinical application. One of the most remarkable breakthroughs in the ischemic conditioning field is pre-/post-conditioning can be performed at a distance or remote from the ischemic target organ because it has the most clinical relevance and translational potential property [5,6]. Remote ischemic postconditioning (RIPostC), which is induced by cycles of a few minutes of I/R applied to a distal artery territory either immediately after the beginning of brain reperfusion or just a few minutes before reperfusion, is an emerging concept for brain I/R injury treatment. However, its potential protective 5
mechanisms have not been well established [7]. Ischemic stroke is a destructive cerebrovascular disease and a leading cause of death and disability worldwide [8]. Although several
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compounds with neuroprotective qualities have been identified in experimental settings, their transition to clinical practice is unsuccessful
RIPostC functions is still urgently needed to be further studied.
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Accumulating evidence indicated that oxidative stress plays a key role in
pathophysiological mechanism of cerebral I/R injury [10,11]. Excessive accumulation of toxic free radicals such as reactive oxygen species (ROS) could trigger diverse signaling pathways and lead to oxidative damage, contributing to the deterioration and progression of ischemic injury after
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[9]. Due to its translational potential property, the mechanism by which
stroke [12]. Superoxide dismutase (SOD), an endogenous anti-oxidant enzyme, plays a crucial role in the oxidative and antioxidative balance to protect organisms from free radical attack. Recent reports showed that ischemic postconditioning attenuates the production of ROS, inhibits malondialdehyde (MDA) increase level [13] and enhances the activity of SOD [14].
Nuclear factor erythroid 2-related factor 2 (Nrf2) is considered to be
a multi-organ protector and is widely deemed to play a vital role in regulating its endogenous antioxidant capacity in cerebral ischemia [15,16]. The antioxidant response element (ARE) is a cis-acting 6
regulatory element that governs the expression of anti-oxidant enzymes and phase II detoxification enzymes. Nrf2 can coordinately regulate the ARE containing genes expression. Differ with other enzymes, heme
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oxygenase 1(HO-1) and quinone oxidoreductase-1(NQO-1) expression is reduced significantly in Nrf2 knockdown mice and is abolished by Nrf2
preconditioning may produce cardioprotection through an enhanced
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HO-1 expression partly via Nrf2 translocation [19].
This study was designed to investigate whether RIPostC, performed by three cycles of occlusion and release of the femoral artery, ameliorated oxidative stress damage in the mouse model of transient middle cerebral artery occlusion (tMCAO), and whether the therapeutic benefit of it was
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disruption [17,18]. It has been reported that delayed remote ischemic
associated with the activation of the Nrf2-ARE pathway. Materials and methods
Experimental animals and grouping Adult male CD1 mice (25 - 30 g) were purchased from the Vital
River Laboratory Animal Technology Co. Ltd, Beijing, China. The experimental procedures were approved by the institutional animal care and use committee and the local experimental ethics committee and conformed to internationally accept ethical standards. All mice were fed a standard diet and water, treated humanely, and maintained at 22±3°C with 12/12 h light/dark cycle, and they were allowed to acclimatize the 7
new surrounding for at least 3 days ahead of any experimentation. Mice were divided into three groups randomly. Sham group: the sham-operated group, in which mice underwent the same surgical
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procedure without inserting a filament and inducing in bilateral hind limbs by occluding and releasing the femoral arteries; the tMCAO group:
group: in which mice were subjected to focal cerebral ischemia and limb
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ischemic postconditioning.
Transient focal cerebral ischemia and remote limb ischemic postconditioning
Transient middle cerebral artery occlusion (tMCAO) surgical procedures were performed as described previously [20]. The tMCAO
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in which mice were subjected to focal cerebral ischemia; the RIPostC
model was induced by right middle cerebral artery occlusion (MCAO) for 1 h. An incision along the vascular route was conducted on femoral triangle of the hind limb, and the bilateral femoral arteries were exposed before MCA occlusion operation. In the RIPostC group, reversible hind limb ischemia was induced in bilateral hind limbs by occluding and releasing the femoral artery for three cycles (occlusion/release 5 min/5 min) immediately after stroke onset. Neurobehavioral evaluation A neurological test was evaluated by an investigator blinded to the experimental groups at 24 h after operation following a modified point 8
scale system based on a 28 point scale of focal neurological scores (FNS) [21], which was based on the following tests: 1, body symmetry, 0 to 4 points; 2, gait, 0 to 4 points; 3, climbing, 0 to 4 points; 4, circling
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behavior, 0 to 4 points; 5, frontal limb symmetry, 0 to 4 points; 6, compulsory circling, 0 to 4 points; 7, whisker response, 0 to 4 points. The
higher score, the more severe impairment of motion injury (the total score
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for each animal is 0 to 28). (n = 15 per group). Measurement of Brain Water Content
Brain water content was measured by the standard wet-dry method at 24 h after operation [22], as described previously [20]. (n = 6 per group).
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7 individual test scores were summed up at the end of the evaluation. The
Infarct volume assessment
Infarct volume was measured at 24 h after reperfusion which was
determined by 2,3,5-triphenyltetrazolium chloride (TTC) [23], as described previously [23]. To offset the effect of brain edema, the corrected infarct volume was calculated as follows: percentage of corrected infarct volume = {[total infarct volume ipsilateral hemisphere
−
−
(the volume of intact
the volume of intact contralateral hemisphere)] /
contralateral hemisphere volume} × 100%. ( 5 slices/mice n = 6 per group). Immunohistochemistry (IHC) 9
Standard Immunohistochemistry procedure was performed as previously described [20] with a modification of antibodies: After blocked in 3% H2O2 and 3% normal goat serum, the brain sections were
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incubated with Nrf2 rabbit polyclonal antibody (1:50, Santa Cruz Biotechnology), HO-1 rabbit polyclonal antibody (1:100, Enzo Life
Technology), in 0.01 mol/L phosphate-buffered saline overnight. Five
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visual fields of ischemic region of the infarct were selected and the immunoreactive cells were counted under a 400 × light microscope. Counting of the positive cells was performed by an investigator blinded to the experimental groups. (n = 3 for each group)
Reverse Transcription-Polymerase Chain Reaction (RT-qPCR)
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Sciences, Inc), and NQO-1 rabbit polyclonal antibody (1:80, Bioworld
Mice (n=6 in each group) were anesthetized and the ischemic cortex
in ipsilateral hemisphere was harvested and frozen in liquid nitrogen at 24 h after reperfusion. As described previously [20], the mRNA expression of Nrf2, HO-1 and NQO-1 were detected by qRT-PCR (MX 3005P, USA) in the presence of a fluorescent dye (SYBR Green I; Cwbio). Forward and reverse primers were 5’-GACAAACATTCAAGCCGATTAGAGG-3’ and
3’-ACTTTATTCTTCCCTCTCCTGCGT-5’ for Nrf2, 5’-TACTCATCCTGAGCTGCTGGT-3’ 3’-TGGACGAAGAAACTCTGTCTGT 10
and for
HO-1,
5’-GGTATTACGATCCTCCCTCAACATC-3’ GAGTACCTCCCATCCTCTCTTCTTC-3’
and for
5’NQO-1,
5’-TGAACGGGAAGCTCACTGG-3’, and
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5’-GCTTCACCACCTTCTTGATGTC-3 for GAPDH. (n = 6 per group) Western Blotting
reperfusion and the ischemic hemisphere of cortex was harvested (from
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an area between 3 and 6 mm frontal to the posterior pole). For the nuclear
protein of Nrf2, the tissue was homogenized in an ice-cold lysis buffer A [10mmol/L
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
[(HEPES), pH 7.8, 10 mmol/L KCl, 2 mmol/L MgCl2, 0.1 mmol/L ethylene diamine tetraacetic acid (EDTA), 1 mmol/L dithiothreitol (DTT),
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All the mice (n=4 in each group) were killed 24 hours after
and 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF)] for 20 min. 10 % Nonidet P-40 (NP-40) was added to the homogenates, and the mixture was centrifuged for 3 min at 10,000 rpm at 4 °C. The supernatant was harvest as a cytosolic fraction for nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, 4-hydroxynonenal (4-HNE) and alpha (IκBα) assays. The pellets were homogenized in ice-cold lysis buffer C (20 mmol/L HEPES, pH 7.8, 300 mmol/L NaCL, 1 mmol/L EDTA, 0.1 mmol/L EGTA, and 0.1 mmol/L PMSF twice) for 40 min. Then centrifuged for 15 min at 14,000 rpm at 4 °C. The supernatant was collected and added PMSF to the final concentration 1 mmol/L as the 11
nuclear protein of Nrf2 (Nrf2,1:100, Santa Cruz), and polyclonal rabbit Lamin B1 antibody (1:1000, Santa Cruz) was used as an internal control. The extracting and analysis of the total protein of HO-1 and NQO-1
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were described as previous reports [20]. Antibodies used here were insteaded by rabbit polyclonal antibody anti-HO-1 (1:500, 1:800, ENZO
(1:10000, Bioworld Technology) (n = 4 in each group) .
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Determination of SOD and MDA levels
Cortical tissues from six mice of each group were collected at 24 hours after operation. The samples were rinsed, weighed, and then homogenized in 9 volumes of 9 g/L ice-cold saline for 10 min using a Dounce Tissue Grinder (Kimble and Kontes, Vineland, NJ, USA).
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antibody), anti-NQO-1, (1:200 Bioworld Technology), and anti-GAPDH
Supernatant homogenate was collected after centrifugation at 4000 rpm/min for 10 min at 4 °C. SOD and MDA levels were measured using an assay kits (A001 and A003; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturers’ instructions. (n = 6 in each group)
Statistical Analysis
All data were analyzed by SPSS 13.0 software, while all quantitative
data were represented as mean ± SD. Statistical comparisons were performed by one-way ANOVA followed by Student-Newman-Keuls test (SNK) for intergroup comparisons. For neurologic deficit scores, 12
Mann-Whitney U test was used for comparisons between groups. P < 0.05 was considered as statistically significant. Results
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RIPostC improved the neurological deficit The Average of neurological score of sham-operated mice was zero.
highest compared with the Sham group (P < 0.05). Compared with
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tMCAO group, there was a significant improvement in neurological function scores in the RIPostC group (P < 0.05) (Figure. 1A). RIPostC reduced the brain edema
Brain water content at 24 hours after operation was shown in Fig. 1B. Ipsilateral Brain water content of Sham group was 78.46% ± 0.77%.
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Whereas the neurological deficit score in the tMCAO group was the
Following ischemia, it shown higher in the tMCAO group ( P < 0.05 vs. Sham). Following RIPostC treatment, as compared with tMCAO group, there was a significant reduction in brain water content in the RIPostC group (81.68% ± 0.66% vs. 84.23 ± 0.63%，P < 0.05). RIPostC reduced infarct volume No infarction was observed in Sham group. Extensive lesion was
found in both striatum and cortex in tMCAO group. Following ischemia, the infarct volume got higher in the tMCAO group ( P < 0.05 vs. Sham). Fig. 1C showing typical photographs of 2% TTC-stained sections from tMCAO and RIPostC-treated mice. The infarct volume was significantly 13
reduced from 42.84% ± 3.98% in tMCAO group to 25.89% ± 3.63% in the RIPostC group ( P < 0.05, Fig. 1D). RIPostC increased the expression of Nrf2, HO-1 and NQO-1 after
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cerebral I/R To investigated the endogenous expression of Nrf2, HO-1 and
western blot and RT-qPCR were performed, respectively. The number of
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positive cells was counted manually and intergroup comparisons were performed. The immunohistochemical staining for Nrf2, HO-1 and NQO-1 of each group was done at 24 h after operation and was shown in Fig. 2A. Few cells were expressing Nrf2, HO-1 and NQO-1 in the cortex of Sham group indicating a low expression in the nonischemic cortex. In
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NQO-1 in ischemic hemisphere of each group, immunohistochemistry,
contrast, the number of positive cells in the ischemic cortex of tMCAO mice was significantly increased compared with Sham group ( P < 0.05). In RIPostC groups, the number of positive cells of Nrf2, HO-1 and NQO-1 was significantly increased than the tMCAO group ( P < 0.05) (Figure. 2B). Results from Western blot analysis and RT-qPCR analysis showed the same patterns at protein levels and the mRNA level of Nrf2, HO-1
and
NQO-1
were
in
according
with
the
results
of
immunohistochemistry ( P < 0.05, Fig. 3A-D). RIPostC increased the activity of SOD and decreased MDA content The activities of SOD were up-regulated and the production of 14
MDA were increased at 24 h after operation in the tMCAO group compared with Sham group ( P < 0.05). Compared with the tMCAO group, we also found that RIPostC can significantly increased the
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activities of SOD and decreased the production of MDA ( P < 0.05). (Figure. 4A,B)
Stroke is a life-threatening disease causing high mortality and
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disability worldwide. Many studies had been proved that RIPostC could
reduce ischemia/reperfusion (I/R) injury and the generation of ROS by protective stimuli in the limb which may be considered as an important trigger involving in the neuroprotection of RIPostC [13]. Our study showed that RIPostC was an effective therapeutic intervention against
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Discussion
cerebral I/R injury in a mouse tMCAO model at 24 h after operation as evidenced by improving neurological outcome, lessening infarct volume and brain edema and exerting anti-oxidant effects. Furthermore, the anti-oxidant effects may be through activating of the Nrf2-ARE pathway. When the oxidative stress occurred, the rapid overproduction of free
radicals overwhelms the detoxification and scavenging capacity of cellular anti-oxidative enzymes like SOD, issuing in a severe damage to neurons [24]. SOD is one of the key free radical scavengers, which detoxifies superoxide radicals to hydrogen peroxide specifically to protect organisms from free radical attack in the body. MDA is one of oxidative 15
stress markers, which expression level reflects the rate and extent of lipid peroxidation as well as the capacity for eliminating free radicals [25]. Compared with the tMCAO group, we also found that RIPostC can
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significantly increase the activities of SOD and decrease the production of MDA in the present study, indicating that the neuroprotection
The overexpression of Nrf2 has become a potential therapeutic
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avenue for ischemic stroke. The antioxidant response element (ARE) is a
cis-acting regulatory element that controlls the expression of phase II detoxification
enzymes
[e.g.
heme
oxygenase-1
(HO-1),
NAD(P)H:quinone oxidoreductase-1 (NQO-1), superoxide dismutase 1 (SOD1), glutathioneS-transferases (GSTs), etc][26]. Nrf2, belonging to
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conferred by RIPostC is due to its anti-oxidative effect.
Cap‘n’collar/basic-leucine zipper family, regulates the ARE containing genes. Numerous antioxidants have been shown to protect the brain from the injury caused by brain ischemia and reperfusion throgh the Nrf2/ARE pathway [27,28]. The chaperone protein HO-1, in concert with cytochrome p450, oxidatively cleaves heme to biliverdin that is subsequently converted to bilirubin. As we all known, both biliverdin and bilirubin have shown anti-oxidative and free radical scavenging properties [29,30]. It is proved that increasing the activity of HO-1 exerts neuroprotective against oxidative and excitotoxic insults relevant to stroke both in vivo and in vitro study [27,31]. NQO-1 is generally 16
considered a detoxifying enzyme because its ability to decrease reactive quinones and quinone imines to less toxic hydroquinonesand less reactive [32]. Various kinds of small molecules like tertiary butyl hydroquinone
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(tBHQ) and sulforaphane, antioxidants are known to activate NQO-1 mediated via ARE [33]. NQO-1 is highly expressed in the lung
antioxidive protection [34]. Our study showed that the expression of Nrf2,
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HO-1 and NQO-1 in ischemic cortex at 24 h after reperfusion were
increased compared with Sham group, and RIPostC significantly increased the expression of Nrf2, HO-1 and NQO-1 compared with tMCAO group, suggesting that RIPostC might have a potent anti-oxidative effect on cerebral I/R through the activation of the
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epithelium and tissues as well as central nervous system that require high
Nrf2-ARE pathway.
It is evident from reports showing that RIPostC is a new
experimented non-pharmacologic intervention and is a potent therapeutic strategy that can be easily and quickly administered to protect the brain
from ischemic damage. However, RIPostC and its fundamental biology are not well understood, and studies to date are largely observational [32,33]. There are plenty of mechanistic work remains to be done not only to provide a theoretical basis for its clinical application, but also to illuminate further pathways involved in the neuroprotection induced by limb ischemic postconditioning. By the identification of those pathways 17
exerted by RIPostC, it will be possible to find out new druggable targets for developing effective strategies to treat ischemic stroke. Our study demonstrated that RIPostC is a new clinically translatable, feasible, and
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safe approach to treat cerebral ischemia, and provided a theoretical basis for its clinical application.
in cerebral I/R due to its anti-oxidative effect and the anti-oxidative effect
Conflict of interest
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may be through activating the Nrf2-ARE pathway.
The authors declared that there are no conflicts of interest.
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In conclusion, our results showed that RIPostC has beneficial effects
Acknowledgments
This work was funded by the Medical Disciplines Funded Projects of Hebei University, China (Grant no. 11ZF006).
18
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Figure legends: Fig. 1. Effect of RIPostC administration on ischemic mice brains. (A) RIPostC improved neurological deficits (n = 15). The neurological deficit
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scores were significantly higher neurological deficit scores in tMCAO group compared with the Sham group and lower in RIPostC group
tMCAO). (B) The water content of ipsilateral hemispheres was higher in
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tMCAO group compared with Sham group and reduced in RIPostC group
compared with tMCAO group (n = 6). ( ☆P < 0.05 vs. Sham; ★P < 0.05 vs. tMCAO). (C) Representative images of TTC staining at brain slices at 24 h after reperfusion (n = 6). The pale region was the infarct brain tissue and the red region was normal. (D) Following ischemia, the infarct volume got higher in the tMCAO group (n = 6). ( ☆P < 0.05 vs. Sham).
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compared with the tMCAO group ( ☆P < 0.05 vs. Sham; ★P < 0.05 vs.
The infarct volume was lessened ( ★P < 0.05 vs. tMCAO)
24
Fig.2. Immunohistochemical staining of Nrf2, HO-1 and NQO-1 in the cerebral cortex at 24 h after reperfusion (n = 3). (A) Representative images for immunoreactive staining of Nrf2, HO-1 and NQO-1 in
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different groups. (400×magnification). (B) Quantification of the number of immunoreactive cells in different groups. The number of
increased in tMCAO and RIPostC. For Nrf2, ☆P < 0.05 vs. Sham; ★P
