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b r a i n r e s e a r c h 1 5 0 3 ( 2 0 1 3 ) 7 8 – 8 8

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nCorrespondenceWai Xi Tou Tiao, Be

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Research Report

Phosphorylation of p38 MAPK mediates hypoxicpreconditioning-induced neuroprotection againstcerebral ischemic injury via mitochondria translocationof Bcl-xL in mice

Li Zhaoa, Xu Liua, Jing Lianga, Song Hana, Yue Wangb, Yanling Yina,Yanlin Luoa, Junfa Lia,n

aDepartment of Neurobiology and Beijing Institute for Brain Disorders, Capital Medical University, Beijing 100069, ChinabDepartment of Neurology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100069, China

a r t i c l e i n f o

Article history:

Accepted 28 January 2013

Hypoxic preconditioning (HPC) initiates intracellular signaling pathway to provide protec-

tion, but the role of p38 mitogen-activated protein kinase (p38 MAPK) in HPC-induced

Available online 8 February 2013

Keywords:

Hypoxic preconditioning

Middle cerebral artery occlusion

Phosphorylation of p38 mitogen-

activated protein kinase

Bcl-xL

Apoptosis

Neuroprotection

nt matter & 2013 Elsevie.1016/j.brainres.2013.01.0

to: Department of Neuroijing 100069, China. Fax:junfali@ccmu.edu.cn (J. L

a b s t r a c t

neuroprotection against cerebral ischemic injuries is a matter of debate. In this study, we

found that HPC could reduce 6 h middle cerebral artery occlusion (MCAO)-induced infarct

volume, edema ratio and cell apoptosis, as well as enhancing the up-regulated p38 MAPK

phosphorylation (P-p38 MAPK) levels in the peri-infarct region of mice after 6 h MCAO.

However, intracerebroventricular injection of p38 MAPK inhibitor SB203580 abolished this

HPC-induced neuroprotection. HPC significantly increased the translocation of anti-

apoptotic Bcl-2-related protein Bcl-xL from the cytosol to the mitochondria in the peri-

infarct region of MCAO mice. Interestingly, the results of reciprocal immunoprecipitation

showed that Bcl-xL and P-p38 MAPK were coimmunoprecipitated reciprocally only in the

peri-infarct region of HPC and MCAO treated mice, while Bcl-xL and total p38 (T-p38

MAPK), not P-p38 MAPK, could be coimmunoprecipited by each other in the brain of

normal control mice. In addition, we found SB203580 significantly decreased P-p38 MAPK

levels, and inhibited HPC-induced mitochondria translocation of Bcl-xL in the brain of HPC

and MCAO treated mice. Taken together, our findings suggested that P-p38 MAPK mediates

HPC-induced neuroprotection against cerebral ischemic injury via mitochondria transloca-

tion of Bcl-xL, which might be a key anti-cell apoptotic mechanism of HPC.

& 2013 Elsevier B.V. All rights reserved.

r B.V. All rights reserved.51

biology and Beijing Institute for Brain Disorders Capital Medical University #10 You An Menþ8610 8395 0060.i).

b r a i n r e s e a r c h 1 5 0 3 ( 2 0 1 3 ) 7 8 – 8 8 79

1. Introduction

Stroke is one of the leading causes of morbidity and mortality in

the world. Among several protection strategies directed against

ischemic injury, hypoxic preconditioning (HPC) demonstrated a

powerful protective potential against ischemic cell death

(Ostrowski et al., 2008; Tsai et al., 2004, 2008). Studies suggested

that mitogen-activated protein kinases (MAPK) had been

involved in this biological process (Heidbreder et al., 2008).

MAPKs are serine–threonine kinases that mediate intracellular

signaling associated with a variety of cellular activities including

cell proliferation, differentiation, survival, death, and transfor-

mation (Dhillon et al., 2007; McCubrey et al., 2006; Torii et al.,

2006). p38 MAPK, a member of MAPK super-family, was activated

by the MAPK kinases 3 and 6 via dual phosphorylation of the

Thr-Gly-Tyr motif in the activation loop (Cohen, 1997; Cuenda

and Rousseau, 2007). Several studies in animal models had

shown that the activation of p38 MAPK increased in ischemic

brain tissue (Irving et al., 2000; Irving and Bamford, 2002; Sugino

et al., 2000). Pretreatment with SB203580, a p38 MAPK inhibitor

may aggravate ischemic brain injury and cerebral vascular

leakage in the model of transient ischemia (Lennmyr et al.,

2003). In this study, we used mouse HPC and middle cerebral

artery occlusion (MCAO)-induced focal cerebral ischemia

models to explore the effect of p38 MAPK on HPC-induced

neuroprotection.

Cell apoptosis has been considered to be one of future

targets for neuroprotective strategies in recent years

(Bielewicz et al., 2010; Ferrer, 2006). Compelling evidences

indicate that apoptosis may occur after transient cerebral

ischemia (MacManus and Buchan, 2000; Islam et al., 1995).

Moreover, recent cerebral ischemia studies in vivo have

revealed that dysregulation of Bcl-2 family proteins can exacer-

bate ischemic neuronal injury and that the interaction between

Bcl-2 family members that suppress (such as Bcl-2 and Bcl-xL)

and those that promote (such as Bax) apoptosis determined

whether cells underwent survival or apoptosis (Billen et al.,

2008; Doeppner et al., 2009; Youle and Strasser, 2008). Bcl-xL was

abundantly expressed in adult neurons and predominantly

localized to the outer mitochondrial membrane and cytosol

(Edlich et al., 2011; Huang and Strasser, 2000; Kaufmann et al.,

2003). Bcl-xL preserved mitochondrial integrity and prevented

the subsequent release of apoptogenic molecules such as

cytochrome c (Gollapudi et al., 2003). In a mouse model,

transcription and expression of Bcl-2 and Bcl-xL were increased

after sublethal forebrain ischemia, which attenuated the DNA

fragmentation induced by lethal ischemia (Wu et al., 2003).

Inhibition of p38 MAPK protected cardiomyocytes from

apoptosis during simulated ischemia in vitro (Cheng, 2006).

Activation of p38 MAPK could protect brain endothelial cells

from apoptosis in stroke (Pfeilschifter et al., 2010). Immuno-

precipitation experiments revealed p38 MAPK-dependent

serine–threonine phosphorylation of Bcl-xL in tumour necrosis

factor (TNF)-treated cells (Sugawara et al., 2004). However,

whether p38 MAPK participate in the HPC-induced neuropro-

tection by reduced neural apoptosis through Bcl-xL remains

unclear. In this study, we explored the role of p38 MAPK-Bcl-xL

antiapoptotic pathway in HPC-induced neuroprotection in the

peri-infarct region of ischemic cortex.

2. Results

2.1. HPC attenuated ischemia-induced injuries in mice

With the increasing times of hypoxia, the tolerance time of

mice increased significantly (n¼30, Fig. 1A). The tolerance

time of hypoxia four times was almost double

(34.0070.80 min) that of hypoxia once (16.9071.20 min). So

mice were exposed to auto-hypoxia for four times in the

following study.

MCAO-induced cerebral ischemic injuries were evaluated

by edema, 2, 3, 5-triphenyltetrazolium chloride (TTC) and

terminal deoxynucleotidyl transferase (TdT)-mediated dUTP

nick end labeling (TUNEL) staining. Fig. 1B showed represen-

tative TTC-stained brain sections from each group. We found

that there was no infarction in the Sham group. A large area

of infarction was seen in the cerebral cortex and striatum in

the Ischemia group (Fig. 1B and C). Similarly, the edema ratio

remarkably increased at 6 h MCAO (Fig. 1D). However, HPC

could significantly decrease the infarct volume and edema

ratio compared with those of the Ischemia group (Fig. 1B–D),

respectively. In concert with these results, the percentage of

apoptotic cells significantly increased in the peri-infarct

region of ischemic cortex but was remarkably reduced by

HPC (Fig. 2).

2.2. Effect of p38 MAPK phosphorylation on HPC-inducedneuroprotection in the ischemic cortex of mice

To reveal the involvement of p38 MAPK in HPC-induced

neuroprotection, we detected the levels of p38 MAPK phos-

phorylation (P-p38 MAPK) and protein expression (T-p38

MAPK) both in the ischemic core and peri-infarct region. As

shown in Fig. 3, we found that the level of P-p38 MAPK in the

peri-infarct region was enhanced in the Ischemia group

compared to that of the Sham group. HPC could further

increase the P-p38 MAPK level in the ischemic peri-infarct

region (Fig. 3). However, there were no significant differences

in the level of P-p38 MAPK between Ischemia and HPCþI

groups whether in the ischemic core or contralateral cortex.

We also found that the level of T-p38 MAPK remarkably

decreased in the ischemic core of Ischemia group. But

combined HPC treatment did not further change T-p38 MAPK

level in the ischemic core as well as peri-infarct region. These

results suggested that p38 MAPK activation by phosphoryla-

tion might be involved in the neuroprotective effect of HPC.

To explore whether p38 MAPK pathway was necessary for

the neuroprotective effect of HPC, we used SB203580, a p38

MAPK inhibitor, before ischemia in this study. We found that

SB203580 treatment in the HPCþI group significantly

increased infarct volume, edema ratio and apoptotic cells at

6 h MCAO compared to DMSO treated controls (Fig. 4A–E).

These results indicated that p38 MAPK inhibitor could abolish

the HPC-induced neuroprotective effects. At the same time,

we found that intracerebroventricular injection of 1 mM

SB203580 significantly inhibited the p38 MAPK phosphoryla-

tion level (Fig. 4F and G).

Fig. 1 – Effects of HPC on edema and infarct volume of mice after MCAO. (A) Statistical results of tolerance time of mice

subjected to auto-hypoxia from 1 to 4 times which were named H1, H2, H3 and H4, respectively (n¼30). �po0.05 compared

with H1. (B) Representative photographs of TTC-stained coronal brain sections of mice from Sham, Ischemia and HPCþI

groups. The white areas were cerebral infarcts. (C) Statistical results of infarct volume from Sham, Ischemia and HPCþI

groups (n¼6 per group). �po0.05 compared with Sham group, #po0.05 compared with Ischemia group, respectively.

(D) Statistical results of edema ratio from Sham, Ischemia and HPCþI groups (n¼6 per group). �po0.05 compared with Sham

group, #po0.05 compared with Ischemia group, respectively.

b r a i n r e s e a r c h 1 5 0 3 ( 2 0 1 3 ) 7 8 – 8 880

2.3. Effect of HPC on Bcl-xL distribution in cytosol andmitochondria in the ischemic cortex of mice

To reveal the involvement of Bcl-xL in HPC-induced neuro-

protection, we observed the level of Bcl-xL in cytosol and

mitochondria. As shown in Fig. 5, we found that Bcl-xL was

localized to both cytosol and mitochondria in the Sham

group. In the ischemic core of cortex, the level of Bcl-xL

decreased in both cytosol and mitochondria significantly at

6 h MCAO and was not affected by HPC. In the peri-infarct

region of cortex, Bcl-xL protein level only decreased in

mitochondrial fraction but did not change in cytosolic frac-

tion at 6 h MCAO. The Bcl-xL level increased remarkably

in mitochondrial fraction, meanwhile decreased significantly

in cytosolic fraction after HPC pretreatment in the ischemic

peri-infarct region (Fig. 5A and B). The ratio of cytosolic

Bcl-xL to mitochondrial Bcl-xL decreased significantly

in the HPCþI group, suggesting that HPC induced the

translocation of Bcl-xL from the cytosol to the mitochondria

(Fig. 5C).

2.4. Co-immunoprecipitation of the T-p38 MAPK or P-p38MAPK with Bcl-xL in the cortex of mice

In order to test whether T-p38 MAPK or P-p38 MAPK inter-

acted with Bcl-xL, we performed the immunoprecipitations

by using the antibodies of anti- Bcl-xL, anti-p38 MAPK and

anti-P-p38 MAPK in the mouse cortex. The results of recipro-

cal immunoprecipitation showed that Bcl-xL could bind T-p38

MAPK in the brain of normal control mice (Fig. 6A). We did

not observe any co-immunoprecipitation of Bcl-xL and P-p38

MAPK in the normal mice (Fig. 6B). But after HPC and MCAO

treatments, the co-immunoprecipitation results actually

showed Bcl-xL could interact with P-p38 MAPK in peri-

infarct region (Fig. 6C). Whole cell lysate and IgG were used

as positive and negative control, respectively (Fig. 6A–C).

Fig. 2 – Effect of HPC on cell apoptosis in the peri-infarct

region after MCAO. (A) Representative photographs of

TUNEL staining showed cell apoptosis in the peri-infarct

region of mice from Sham, Ischemia and HPCþI groups.

TUNEL positive cells were green, while Hoechst positive

cells were blue (scale bar¼50 lm). (B) Statistical results of

TUNEL staining from Sham, Ischemia and HPCþI groups

(n¼6 per group). npo0.05 compared with Sham group,

#po0.05 compared with Ischemia group, respectively.

Fig. 3 – Effects of HPC on p38 MAPK phosphorylation and

protein expression in the cortex of mice. (A) Representative

results of Western blot showed the levels of P-p38 MAPK

and T-p38 MAPK in the cortex from Sham, H4, Ischemia and

HPCþI groups. (B) Quantitative analysis showed the

changes of P-p38 MAPK level and T-p38 MAPK level in the

cortex of mice (n¼6 per group). The total level of p38 MAPK

was normalized against the b-actin and the level of

phosphorylated p38 MAPK was normalized against the total

p38 MAPK. I, ischemic core; P, peri-infarct region; C,

contralateral cortex to ischemic hemisphere. npo0.05

compared with Sham group, #po0.05 compared with P of

Ischemia group, respectively.

b r a i n r e s e a r c h 1 5 0 3 ( 2 0 1 3 ) 7 8 – 8 8 81

2.5. Bcl-xL translocation was associated with p38 MAPKactivation in the ischemic cortex of mice after HPC

To explore whether Bcl-xL translocation was associated with

p38 MAPK activation, we further observed the effect of

SB203580 on Bcl-xL mitochondria translocation in the peri-

infarct region of ischemic cortex. We found that the protein

level of Bcl-xL in HPCþSB203580þI group decreased remark-

ably in mitochondrial fraction and increased significantly in

cytosolic fraction compared with DMSO treatment (Fig. 7A

and B). The ratio of cytosolic Bcl-xL to mitochondrial Bcl-xL

increased remarkably in the HPCþSB203580þI group, suggest-

ing that SB203580 partly blocked the mitochondria transloca-

tion of Bcl-xL induced by HPC in the peri-infarct region

(Fig. 7C).

3. Discussion

Among several protection strategies directed against ischemic

injury, hypoxic/ischemic preconditioning demonstrated a

powerful protective potential against ischemic cell death

(Bolli, 2007). Since pretreatment with ischemia is not applic-

able to patients, many attempts have been undertaken to

achieve the protective effect of ischemic preconditioning

either pharmacologically (Zhao and Kukreja, 2002) or by

pretreatment with moderate environmental stress, e.g., HPC.

HPC initiates several intracellular signaling pathways to pro-

vide protection against subsequent cerebral ischemic injuries

(Long et al., 2006; Zhang et al., 2007, 2009, 2011). Recently, the

concept of two proposed types of HPC has been widely

accepted (Tsai et al., 2004). The early preconditioning which

occurred within minutes and lasted approximately 2 h after

HPC was reported to be related to changes in the intracellular

signal transduction pathways, such as the activation of protein

Fig. 4 – Effects of SB203580 on infarct volume, edema and cell apoptosis in the cortex of mice. (A) Representative photographs

of TTC-stained coronal brain sections from HPCþDMSOþI and HPCþSB203580þI groups. The white areas were cerebral

infarcts. (B) Statistical results of infarct volume from HPCþDMSOþI and HPCþSB203580þI groups (n¼6 per group). (C)

Statistical results of edema ratio of mice from HPCþDMSOþI and HPCþSB203580þI groups (n¼6 per group). (D)

Representative photographs of TUNEL staining showed cell apoptosis in the peri-infarct region of mice from HPCþDMSOþI

and HPCþSB203580þI groups. TUNEL positive cells were green, while Hoechst positive cells were blue (scale bar¼50 lm).

(E) Statistical results of TUNEL staining from HPCþDMSOþI and HPCþSB203580þI groups (n¼6 per group). (F) Representative

results of Western blot showed the levels of P-p38 MAPK and T-p38 MAPK in the peri-infarct region from HPCþDMSOþI and

HPCþSB203580þI groups. (G) Quantitative analysis showed the changes of P-p38 MAPK level in the peri-infarct region of

mice (n¼4 per group). The level of P-p38 MAPK was normalized against the b-actin. �po0.05 compared with

HPCþDMSOþI group.

b r a i n r e s e a r c h 1 5 0 3 ( 2 0 1 3 ) 7 8 – 8 882

kinases and phosphatase (Meldrum et al., 1997). However, the

delayed preconditioning appeared 24–72 h after the initial

preconditioning stimulation, and the mechanisms were

reported to be associated with the activation of cellular

genome changes and protein synthesis processes. Ischemic

preconditioning, 24 h prior to the 120 min MCAO, decreased

significantly the infarct volumes (Taskapilioglu et al., 2009).

Intermittent hypoxia intervention for 7 days after 7 days of

recovery post ischemia could reduce the infract volume, and

did not increase the mortality rate (Tsai et al., 2008). In the

present study, We used a unique HPC-induced ‘‘auto-hypoxia’’

mouse model that mimicked clinical asphyxia (Li et al., 2005)

Consistent with the above results, we found HPC also

remarkably decreased infarct volume, edema ratio and neural

apoptosis induced in 6 h MCAO model in mice. These findings

indicate that HPC had protective effects on cerebral ischemia.

P38 MAPK was activated in response to stress signals and

focal cerebral ischemia (Irving et al., 2000). The immunohis-

tochemical study showed that active p38 MAPK immunor-

eactivities were enhanced at 15 min of reperfusion and then

gradually reduced and disappeared in the hippocampal CA1

region (Sugino et al., 2000). In CA3 neurons, active p38 MAPK

immunoreactivities were enhanced at 15 min of reperfusion

and peaked at 6 h of reperfusion and then gradually reduced

but was continuously detected 72 h after ischemia (Sugino

et al., 2000). Phospho-p38 MAPK immunostaining was

Fig. 5 – Effects of HPC on Bcl-xL protein expression in

cytosol and mitochondria of cortex. (A) Representative

results of Western blot showed Bcl-xL protein expression in

cytosol and mitochondria in the cortex from Sham, H4,

Ischemia and HPCþI groups. (B) Quantitative analysis

showed the changes of Bcl-xL distribution in cytosol and

mitochondria in the cortex of mice (n¼6 per group). The Bcl-

xL level in cytosol was normalized against the b-actin level.

The Bcl-xL level in mitochondria was normalized against

the COX-IV level. (C) Quantitative analysis showed the ratio

of cytosolic Bcl-xL to mitochondrial Bcl-xL. �po0.05

compared with Sham group, #po0.05 compared with P of

Ischemia group, respectively.

Fig. 6 – Co-immunoprecipitation of the T-p38 MAPK or P-p38

MAPK with Bcl-xL in the cortex of mice.

(A) Immunoprecipitation assay showed that the association

between Bcl-xL and T-p38 MAPK existed in the cortex of

normal mice. (B) Immunoprecipitation assay showed no co-

immunoprecipitation of the P-p38 MAPK with Bcl-xL in the

cortex of normal mice. (C) Immunoprecipitation assay

showed that the association between Bcl-xL and P-p38

MAPK existed in the peri-infarct region from HPCþI group.

WCL and IgG were used as positive and negative control,

respectively. WCL, whole cell lysate.

b r a i n r e s e a r c h 1 5 0 3 ( 2 0 1 3 ) 7 8 – 8 8 83

markedly increased in cells with astrocyte-like morphology in

both ischemic core and penumbra-like regions (Irving et al.,

2000). In this study, immunoblot analysis demonstrated that T-

p38 MAPK level decreased significantly at 6 h MCAO in the

ischemic core of cortex. More neural cell necrosis in the ischemic

core might contribute to these results. On the other hand, in the

ischemic peri-infarct region, the level of P-p38 MAPK increased

significantly but the T-p38 MAPK level did not change.

Moreover, several reports found that p38 MAPK could be

implicated in the preconditioning effects (Moolman et al.,

2006; Sun et al., 2006, 2010; Yamashita et al., 2009). In the

apoptosis model using global ischemia, ischemic precondi-

tioning resulted in a significant decrease in p38 MAPK

activation at the end of reperfusion when compared to non-

preconditioned isolated rat hearts (Moolman et al., 2006).

Expression of P-p38 MAPK was increased after limb ischemic

preconditioning in the CA1, CA3 and dentate gyrus regions of

hippocampus (Sun et al., 2006). The level of P-p38 MAPK was

up-regulated at 6 h and reached its peak 12 h in the CA1

region of the hippocampus after limb ischemic precondition-

ing (Sun et al., 2010). However, the pretreatment of

3.5-atomsphere absolute (3.5 ATA) and hyperbaric oxygen

significantly decreased the level of P-p38 MAPK at 10-min

reperfusion (Yamashita et al., 2009). In the present study, we

found that HPC could further increase the level of P-p38

MAPK in the ischemic peri-infarct region, but not affect the

T-p38 MAPK level both in the ischemic core and peri-infarct

Fig. 7 – Effects of SB203580 on Bcl-xL protein expression in

cytosol and mitochondria of cortex. (A) Representative results

of Western blot showed Bcl-xL protein expression in cytosol

and mitochondria in the cortex from HPCþDMSOþI and

HPCþSB203580þI groups. (B) Quantitative analysis showed the

changes of Bcl-xL protein level in cytosol and mitochondria

after SB203580 or DMSO administration (n¼6 per group).

The Bcl-xL protein level in cytosol was normalized against the

b-actin. The Bcl-xL protein level in mitochondria was

normalized against the COX-IV. (C) Quantitative analysis

showed the ratio of cytosolic Bcl-xL to mitochondrial Bcl-xL

after SB203580 or DMSO administration. �po0.05 compared

with P of HPCþDMSOþI group.

b r a i n r e s e a r c h 1 5 0 3 ( 2 0 1 3 ) 7 8 – 8 884

region. These results suggested that p38 MAPK phosphoryla-

tion, not expression level, involved in the HPC-induced

neuroprotection.

However, the role of p38 MAPK in preconditioning effect

has not been defined unequivocally. SB203580 was first

described as an inhibitor of p38MAPK activity by competing

with ATP for binding (Young et al., 1997). However, it was later

demonstrated that SB203580 also prevented p38MAPK

phosphorylation (Galan et al., 2000). In this study we really

observed that intracerebroventricular injection of 1 mM

SB203580 significantly inhibited p38MAPK phosphorylation.

SB203580 could reduce preconditioning effects of sublethal-

ischemia, isoflurane-exposure, or limb-ischemia (Sun et al.,

2006; Zheng and Zuo, 2004). Several reports suggested that

the cardioprotective effects of preconditioning could be

abolished by SB203580 (Fryer et al., 2001; Nakano et al.,

2000; Sato et al., 2000). SB203580 blocked the protective effect

of limb ischemic preconditioning against delayed neuronal

death that was normally induced by lethal brain ischemic

insult (Sun et al., 2010). But Lochner et al. (2003) found that in

the isolated perfused rat heart improved functional recovery

and reduction in infarct size were associated with attenua-

tion of p38 MAPK activation by SB203580 during sustained

ischemia-reperfusion. SB203580, when given between admin-

istration of 3.5 ATA and hyperbaric oxygen treatment,

remarkably increased the number of survived neurons

(Yamashita et al., 2009). Bell et al. (2008) reported that

SB203580 blocked p38 MAPK activity, but did not block

ischemic preconditioning and was not protective when given

during ischemia in rat heart . The different effects of

SB203580 may be attributable to the use of different species,

models, protocols and measured end-points. Using our estab-

lished mouse HPC and MCAO models of mice, we found that

SB203580 could abolish the neuroprotective effects induced

by HPC. Taken together, our results suggested that HPC might

attenuate MCAO-induced cerebral ischemic injuries of mice

through the phosphorylation of p38 MAPK.

The ischemic penumbra or peri-infarct region is poten-

tially salvageable and thus represents an opportunity for

therapeutic intervention although it is under threat of infarc-

tion. Apoptosis is believed to contribute to loss of neurons in

the ischemic penumbra. Bcl-xL is a potent inhibitor of

programmed cell death and acts by heterodimerization with

other Bcl-2 family members to inhibit caspase activation,

presumably by blocking release of cytochrome c (Antignani

and Youle, 2006). Bcl-xL promoted cell survival by inhibiting

the function of the proapoptotic Bcl-2 proteins (Chipuk and

Green, 2008; Youle and Strasser, 2008). Bcl-xL protected lung

endothelial cells against hypoxia/reoxygenation-induced cell

death by blocking Bax and Bid mitochondrial translocation

and inhibiting mitochondrial cytochrome c release. A large

number of studies have shown that HPC attenuated apoptosis

through changing the expression of Bcl-xL (Rybnikova et al.,

2006; Wang et al., 2010). HPC protected neurons in rat

hippocampus and neocortex from the post-hypoxic apoptotic

transformations via persistent overexpression of Bcl-xL

(Rybnikova et al., 2006). HPC suppressed group III secreted

phospholipase A2-induced apoptosis via Janus-activated

kinase 2/signal transducers and activators of transcription 3

signal pathways that regulated the expression of Bcl-xL in

cultured cortical neurons (Wang et al., 2010). In the present

study, we found that HPC triggered Bcl-xL translocation to

mitochondria from cytosol in the peri-infarct region of

b r a i n r e s e a r c h 1 5 0 3 ( 2 0 1 3 ) 7 8 – 8 8 85

ischemic cortex. Our results suggested that Bcl-xL in mito-

chondria might participate in antiapoptosis effect induced

by HPC.

Various upstream signaling mechanisms can stimulate

transcription of Bcl-xL or suppress the inhibitory effect of

Bad on Bcl-xL, such as Janus-activated kinase 2, extracellular

signal-regulated kinase 1/2, Akt, and protein kinase A path-

ways (Basu and Haldar, 2003; Kharbanda et al., 2000; Saito

et al., 2003; Sugawara et al., 2004; Upreti et al., 2008). P38

MAPK was found to play a crucial role in the ischemic

preconditioning-induced cardioprotection through activation

of Bcl-2 (Kilic et al., 2005). P38 MAPK could mediate

TNF-induced apoptosis in endothelial cells via phosphoryla-

tion and down-regulation of Bcl-xL (Saito et al., 2003). In

this study, we found that Bcl-xL and T-p38 MAPK could

be coimmunoprecipited by each other in the cortex of

normal control mice. We did not observe any co-

immunoprecipitation of Bcl-xL and P-p38 MAPK in the nor-

mal group. But Bcl-xL and P-p38 MAPK were coimmunopre-

cipitated reciprocally in the peri-infarct region of HPC and

MCAO treated mice possibly because of the quadrupled P-p38

MAPK level. Using SB203580, the effect of HPC on mitochon-

dria translocation of Bcl-xL was inhibited: the protein level of

Bcl-xL decreased remarkably in mitochondrial fraction but

increased significantly in cytosolic fraction.

In summary, our findings suggested that P-p38 MAPK

mediates HPC-induced neuroprotection against cerebral

ischemic injury via mitochondria translocation of Bcl-xL,

which might be a key anti-apoptotic mechanism of HPC.

Further studies about the Bcl-xL phosphorylation sites by p38

MAPK during cerebral ischemia and HPC and the relations

between Bcl-xL phosphorylation and translocation will be

required.

4. Experimental procedures

4.1. Animals

The experimental procedures were carried out in accordance

with the Chinese regulations involving animal protection and

approved by the animal ethics committee of the China

Capital Medical University (Permit Number: 2011-X-026).

Experiments were performed at room temperature

(20–22 1C) on male BALB/c mice (12–14 W, 18–22 g). All surgery

was performed under sodium pentobarbital anesthesia, and

all efforts were made to minimize suffering. Animals were

randomly divided into six groups, namely Sham group (no

MCAO and HPC treatment), H4 group (auto-hypoxia four

times), Ischemia group (MCAO-induced cerebral ischemia),

HPCþI group (ischemia with H4 treatment), HPCþDMSOþI

group (ischemia with HPC and DMSO treatments) and

HPCþSB203580þI group (ischemia with HPC and SB203580

treatments).

4.2. Animal models

The auto-hypoxia-induced HPC mouse model was prepared

as our reported (Li et al., 2005; Niu et al., 2005). In brief, mice

were placed individually in a 125 ml airtight jar, which was

full of fresh air and sealed with a rubber plug to duplicate a

progressive auto-hypoxia environment. Mice were removed

as soon as the first signs of gasping appeared. A minimum of

30 min was allowed for recovery in normoxic condition, and

then the mice were switched to another hermetically sealed

jar of the same volume. All of the HPC mice underwent 4

times repetitive hypoxic exposures. It was designated as

H1–H4 when the mice were exposed to hypoxia once, twice,

three time and four times, respectively.

MCAO was performed 1 h after pretreatment of HPC. The

MCAO-induced cerebral focal ischemia mouse model was

conducted as described previously with minor modification

(Chen et al., 2007). An arteriotomy was made in common

carotid artery allowing the introduction of a 5-0 surgical

filament with its tip (0.23 mm in diameter) rounded by heat

(#1622-A, Shadong Biotechnology Company, Beijing, China).

The filament was gently advanced into the internal carotid

artery to occlude the middle cerebral artery. In the Sham

group, the mice received the same surgical exposure of

carotid artery without occlusion. During the surgery, body

temperature was maintained with the use of a heating lamp

and thermal blanket.

4.3. Drug treatment

Just before MCAO, the mice received an intracerebroventri-

cular injection of 5 ml SB203580 (1 mM, #S8307-1MG, Sigma-

Aldrich, St. Louis, MO, USA) or DMSO (0.1%) in the ischemic

side. Drug administration procedures followed the method

described by Munoz et al. (2003). The location of each

injection was 0.5 mm posterior to the bregma, 1.0 mm lateral

to the midline, and 3.5 mm below the skull surface. The mice

underwent MCAO as described above.

4.4. Measurement of infarct size, edema ratio and TUNELstaining

Infarct volume, edema ratio and cell apoptosis were assessed

at 6 h after MCAO, although at this time point cerebral

ischemic injuries were still under development, ischemic

penumbra or peri-infarct region exited more apparently and

the p38 MAPK mediated molecular changes in this region at

early stage could be studied.

Mice were sacrificed at 6 h after MCAO, and then the brain

was quickly removed and cut into 1.5 mm-thickness coronal

sections. Brain sections were incubated for 20 min in a

solution of 0.5% TTC in 10 mM phosphate buffered saline at

37 1C, and then the slices were photographed by using

scanner. According to ischemic volume evaluation procedure

reported by Wexler et al. (2002), the infarct size was analyzed.

Edema ratio (E) was calculated using the following equation:

E¼ (SVL�SVR)/(SVLþSVR)�100%. SVL and SVR are the

volume of left (ischemic) and right (non-ischemic) hemi-

sphere volume, respectively.

Mouse brains were cut into 10 mm-thickness sections for

TUNEL staining. The TUNEL staining kit (#G3250, DeadEndTM-

Fluorometric TUNEL system, Promega, Madison, WI, USA)

was used to assess cell apoptosis. In brief, the brain sections

were placed in equilibration buffer and incubated with

nucleotide mix and rTdT enzyme at 37 1C for 1 h. Using 2X

b r a i n r e s e a r c h 1 5 0 3 ( 2 0 1 3 ) 7 8 – 8 886

saline–sodium citrate buffer stopped the reaction. Hoechst

33258 was used to stain neural nuclei. The images were

visualized by fluorescence microscope (Leica TCS SP5, Wet-

zlar, Germany). The TUNEL positive cells in peri-infarct region

of cortex were counted.

4.5. Sample preparation and Western blot analysis

As previously reported (Jiang et al., 2009; Li et al., 2005; Zhang

et al., 2011), the tissues from ischemic core (I), peri-infarct

region (P) and contralateral cortex (C) were collected. The

frozen samples were rapidly thawed and homogenized at 4 1C

in buffer A (5 mM Tris–Cl, pH 7.5, containing 2 mM DTT, 2 mM

EDTA, 1 mM EGTA, 5 mg/ml each of leupeptin, aprotinin,

pepstatin A and chymostatin, 50 mM potassium fluoride,

50 mM okadaic acid, 5 mM sodium pyrophosphate). Homoge-

nates were centrifuged at 30,000g for 30 min at 4 1C.

The supernatants were collected as cytosolic fraction. The

mitochondrial fraction was obtained from the pellets accord-

ing to manufacture instruction of mitochondria isolation kit

(#GMS10006.2, GENMED SCIENTIFICS INC, Boston, MA, USA).

In order to get whole tissue homogenate, frozen samples

were homogenized at 4 1C in buffer C [(Buffer A mixed with

2% sodium dodecyl sulfate (SDS)] and sonicated to dissolve

the tissue completely. The protein concentration was deter-

mined by BCA kit (Pierce Company, Rockford, IL, USA) with

bovine serum albumin diluted in lysis buffer as standard.

The tissue extracts with equal amount of total protein

were separated by 10% SDS polyacrylamide gel electrophor-

esis (SDS-PAGE) and the proteins were transferred to poly-

vinylidene difluoride membranes, and probed with anti-

phospho-p38 MAPK (#P1491-1VL, Sigma-Aldrich, St. Louis,

MO, USA), anti-p38 MAPK (#9212, Cell Signalling Technology,

Danvers, MA, USA) and anti-Bcl-xL (#2764, Cell Signaling

Technology, Danvers, MA, USA) antibodies, respectively.

The horseradish peroxidase-conjugated goat anti-rabbit or

anti-mouse IgG (Stressgen Biotechnologies Corporation, Vic-

toria, BC, Canada) was used as second antibodies. Following

incubation with the primary and secondary antibodies, the

Enhanced Chemiluminescence kit (Pierce Company, Rock-

ford, IL, USA) was employed to detect the signals. To verify

equal loading of protein, the blots were reprobed with

primary monoclonal antibodies against b-actin (#60008-1-Ig,

Proteintech Company, Chicago, IL, USA) or COX-IV (#11242-1-

AP, Proteintech Company, Chicago, IL, USA).

4.6. Coimmunoprecipitation

Nondenaturing lysis buffer extracted 500 mg protein of mouse

cortex was immunoprecipitated with 2 mg of Bcl-xL antibody,

p38 MAPK antibody, P-p38 MAPK antibody and IgG, respec-

tively (Bu et al., 2011). After 3 h incubation, protein

G sepharose was added for 12 h at 4 1C and centrifuged for

1 min at 12,000g. The precipitates were rinsed with IP buffer

(0.5% NP-40, Tris–Cl pH 8.0, 0.15 M NaCl) four times to remove

nonspecific interaction products. The immunoprecipitates

were resolved in loading buffer for SDS-PAGE.

4.7. Statistics

Quantitative analysis for immunoblotting was done by

Quantitative-One software (Gel Doc 2000 imaging system,

Bio-Rad Company, CA, USA). For protein level, the ratio of p38

MAPK or Bcl-xL (band density of protein/band density of

b-actin or COX-IV) was expressed as 100% in the control

group. Both bands of Bcl-xL were quantitative analyzed. The

data from other groups were expressed as percentage of that

from control group. For p38 MAPK phosphorylation, the ratio

of P-p38 MAPK to T-p38 MAPK in sham group was recognized

as 100%, and the values of ischemic groups were expressed as

a percentage of sham group. The presented values were

expressed as mean7SE. Statistical analysis was conducted

by one-way analysis of variance (ANOVA) followed by

Student–Newma–Keuls post hoc analysis. In all cases,

po0.05 was considered statistically significant.

Acknowledgments

This work was supported by the following grants: National

Natural Science Foundation of China (31071048, 31171147 and

31140005), China 973 Pre-program (2011CB512109), Ph.D.

Programs Foundation of Ministry of Education of China

(20091107110001), Basic and clinical research joint program

of Capital Medical University (11JL34 and 11JL44), Research

fund of Capital Medical University (2011ZR02) and the Scien-

tific Research Foundation for the Returned Overseas Chinese

Scholars, State Education Ministry. The funders had no role in

study design, data collection and analysis, decision to pub-

lish, or preparation of the manuscript.

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