Accepted Manuscript

Propofol prevents cerebral ischemia-triggered autophagy activation and cell

death in the rat hippocampus through the NF-κB/p53 signaling pathway

Derong Cui, Li Wang, Wei Jiang, Aihua Qi, Quanhong Zhou, Xiaoli Zhang

PII: S0306-4522(13)00387-4

DOI: http://dx.doi.org/10.1016/j.neuroscience.2013.04.054

Reference: NSC 14584

To appear in: Neuroscience

Accepted Date: 26 April 2013

Please cite this article as: D. Cui, L. Wang, W. Jiang, A. Qi, Q. Zhou, X. Zhang, Propofol prevents cerebral ischemia-

triggered autophagy activation and cell death in the rat hippocampus through the NF-κB/p53 signaling pathway,

Neuroscience (2013), doi: http://dx.doi.org/10.1016/j.neuroscience.2013.04.054

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Title Page

(i) Article Title:

Propofol prevents cerebral ischemia-triggered autophagy activation and cell death in the rat hippocampus through

the NF-κB/p53 signaling pathway

(ii) Author names:

Derong Cui †, Li Wang †, Wei Jiang, Aihua Qi, Quanhong Zhou, Xiaoli Zhang, († these authors contributed equally

to this work)

(iii) Academic ranks and institutional affiliations:

Derong Cui, M.D., Department of Anesthesiology, Shanghai Sixth People's Hospital Affiliated with Shanghai

Jiaotong University.

Li Wang, M.D., Professor, Department of Anesthesiology, Shanghai Sixth People's Hospital Affiliated with

Shanghai Jiaotong University.

Wei Jiang, M.D. Ph.D., Professor, Department of Anesthesiology, Shanghai Sixth People's Hospital Affiliated

with Shanghai Jiaotong University.

Aihua QI, M.D., Department of Anesthesiology, Shanghai Sixth People's Hospital Affiliated with Shanghai

Jiaotong University.

Quanhong Zhou, M.D., Department of Anesthesiology, Shanghai Sixth People's Hospital Affiliated with Shanghai

Jiaotong University.

Xiaoli Zhang, M.D., Department of Anesthesiology, Shanghai Sixth People's Hospital Affiliated with Shanghai

Jiaotong University.

(iv) Corresponding author information:

Corresponding author name: Wei Jiang

Complete mailing address: NO.600, Yi Shan RD., Shanghai, China, 200233

Telephone number: +86 21 64369181 58328

Facsimile number: +86 21 64369181 58330

E-mail address: jiangw@sjtu.edu.cn

(v) Department/institution affiliated with this work:

Department of Anesthesiology, Shanghai Sixth People's Hospital Affiliated with Shanghai Jiaotong University.

(ⅵ) Acknowledgements

The work was supported by grants from the Natural Science Foundation of Shanghai Jiaotong University

(2012043), the Ph.D. Programs Foundation of the Ministry of Education of China (20120073110087) and the Ph.D.

Innovation Fund Project to Dr. Cui from Shanghai Jiaotong University (BXJ201237). The funders had no role in

study design, data collection and analysis, decision to publish, or preparation of the manuscript.

(vii) Abbreviations:

H/I, hypoxia-ischemia; I/R, ischemia-reperfusion; 3-MA, 3-methyladenine; DMSO, dimethylsulfoxide;

DRAM, damage-regulated autophagy modulator; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LC3,

microtubule-associated protein 1 light chain 3; NF-κB, nuclear factor-kappa B; PBS, phosphate-buffered saline;

PCR, polymerase chain reaction; PFT-α, Pifithrin-alpha; PUMA, p53-upregulated modulator of apoptosis; SDS,

sodium dodecyl sulfate; TBST, Tris-buffered saline containing 0.2% Tween-20;

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(ⅷ) Key words:

autophagy, propofol, cerebral ischemia, NF-κB, p53, LC3

Abstract

Propofol (2,6-diisopropylphenol) has been shown to attenuate neuronal injury under a number of experimental

conditions; however, the mechanisms involved in its neuroprotective effects remain unclear. We therefore

investigated whether inhibition of p53 induction by propofol contributes to the neuroprotection of cerebral

ischemic cell death through both autophagic and apoptotic mechanisms. A transient global cerebral

ischemia-reperfusion (I/R) model was produced with a 10-minute, 2-vessel occlusion. The change in target genes

including damage-regulated autophagy modulator (DRAM), microtubule-associated protein 1 light chain 3 (LC3),

Beclin 1, cathepsin D, cathepsin B, p53-upregulated modulator of apoptosis (PUMA), Bax and Bcl-2 upon p53

inhibition was assessed with the co-administration of the intravenous anesthetic propofol and 3-methyladenine

（3-MA）, Pifithrin-alpha （PFT-α） or SN50. The I/R-induced increases of protein levels of p53 and LC3-Ⅱ were

significantly inhibited by treatment with propofol, 3-MA or PFT-α. The I/R-induced increases of protein levels of

DRAM, Beclin 1, active cathepsin D and cathepsin B were significantly inhibited by treatment with propofol,

PFT-α or SN50. The negative effects of the I/R-induced up-regulation of PUMA and Bax and the down-regulation

of Bcl-2 in the rat hippocampus were all blocked by treatment with propofol, PFT-α or SN50. Our results suggest

that cerebral ischemia-reperfusion can induce NF-κB-dependent expression of p53. The autophagic and apoptotic

mechanisms participate in programmed cell death by regulating the p53-mediated pathway. Our results are the first

to show that propofol, at clinically relevant concentrations, attenuated cell death through both autophagic and

apoptotic mechanisms in the rat hippocampus after a cerebral I/R insult.

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Introduction

The clinical efficacy of propofol for the induction and maintenance of anesthesia has been clearly

demonstrated in many literature reports. In addition to its use as a sedative-hypnotic, propofol displays

neuroprotective effects (Young et al., 1997). In an experimental cerebral ischemic model in animals, propofol

treatment was shown to improve neurological outcome and decrease infarction size in the brain (Ergün et al.,

2002). These beneficial effects on the neuronal system have been suggested to involve the activation of the

γ-aminobutyric acid–A (GABAA) receptor, Ca2+ channel inhibition and the scavenging of free radical species.

Propofol administration also ameliorates autophagy in ischemic hippocampal neurons. At the cellular and

molecular levels, treatment of hypoxic neuronal PC12 cells and the rat hippocampus after ischemic attack with

propofol inhibits activation of autophagy-related proteins including phosphatidylinositol 3 kinase class Ⅲ (PI3K

class Ⅲ), Beclin-1 and microtubule-associated protein 1 light chain 3-Ⅱ(LC3-II), with augmented neuronal

survival (Cui et al., 2012). Although autophagy has been demonstrated to remove potentially hazardous and toxic

substances from sensitive tissue, it can also promote autophagic cell death, termed programmed cell death type II

(Cui et al., 2012; Yousefi et al., 2007). Autophagic cell death is distinguished by the aggregation of autophagic

vacuoles. Evidence suggests that autophagy is involved in the execution of hypoxia-ischemia (H/I)

injury-induced neuronal death in the adult hippocampus. It has been reported that cerebral ischemia-reperfusion

results in the activation of autophagy (Cui et al., 2012; Chu, 2006; Wen et al., 2008; Luo et al., 2011). Blocking

autophagy with 3-methyladenine (3-MA) or LY294002 partially inhibits cerebral-ischemic injury, suggesting that

autophagy results in ischemic damage of hippocampal neurons (Cui et al., 2012). Therefore, therapeutic strategies

to suppress autophagy-induced neuronal death may be helpful in the treatment of both pediatric and adult H/I

brain injury (Koike et al., 2008).

The molecular biochemical mechanisms concerned with cell death remain largely unexplored. There is

increasing evidence demonstrating a complex interplay between autophagy and apoptosis (Perlman et al., 2008;

Komatsu et al., 2005; Kuma et al., 2004). Several regulators in the apoptotic pathway also function as modulators

of autophagy activation (Shintani et al., 2004; Zhu et al., 2007; Nixon et al., 2006). Recent experiments have

demonstrated that quinolinic acid-induced neuronal autophagy and apoptosis involve the activation of NF-κB and

the induction of the pro-apoptotic protein p53. Inhibition of NF-κB or p53 has strong neuroprotective effects that

involve the down regulation of autophagic and apoptotic molecules (Wang et al., 2009). Recently, it has been

reported that the inhibition of NF-κB or p53 induces a strong neuroprotection after neonatal hypoxia-ischemia

(Nijboer et al., 2008). As cerebral ischemia-reperfusion (I/R) is common in the peri-operative period (Li et al.,

2010), we speculate that the NF-κB-regulated p53 pathway may contribute to I/R neuronal injury by activating

autophagy and apoptosis. Moreover, it will be of interest to identify whether intravenous propofol could suppress

the NF-κB-dependent expression of p53, thus inhibiting the cell death induced by cerebral I/R injury. Here, we

investigate how this anesthetic drug influences pyramidal neuron cell death induced by cerebral I/R injury in the

rat hippocampus. We attempted to explore the possible mechanisms of propofol action in the neuroprotection of

pyramidal neuron cell death, a type of cellular damage that occurs during the I/R process.

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Experimental procedures

Animal and surgical protocols

Male Sprague-Dawley rats (weighing 250–300 g) were supplied by the Experimental Animals Center of

Shanghai Jiaotong University. All procedures, including the I/R injury model as described in our previous study

(Cui et al., 2012), were in accordance with the Guide for the Care and Use of Laboratory Animals (Guide for the

Care and Use of Laboratory Animals, published by the National Institutes of Health 1996) and approved by the

Animal Research Committee of Shanghai Jiaotong University. All efforts were made to minimize animal suffering

and reduce the number of animals used.

After fasting for 8–12 h, anesthesia was administered in a Plexiglas chamber with 4% halothane followed by

tracheal intubation, and the rats were ventilated with 1.5% halothane in 30% O2/70% N2O. Muscle relaxants were

not administered during the anesthesia. The animal preparations have been described in detail in our previous

report (Cui et al., 2012). The left femoral artery was cannulated to monitor the blood pressure and collect the blood,

and the right external jugular vein was used for drug administration and blood reinjection. Digital thermistor

probes (Multi-thermistor Meter D321; Technol Seven, Yokohama, Japan) were placed in the rectum to monitor the

core temperature, which was maintained at 37±0.5°C using an electrically heated blanket. The arterial blood

samples were collected for blood gas analysis after the isolation of the bilateral common carotid arteries from the

carotid sheaths using a ventral midline incision. If the blood gas parameters were PO2 90–140 mmHg, PCO2 35–45

mmHg, pH 7.35–7.45, GI 150-180 mg/dl, cerebral ischemia was induced by clamping the common carotid arteries

with small vascular clips and inducing hypotension (MAP: 40±5 mmHg) by withdrawing and injecting blood for

10 min. Forebrain ischemia was confirmed with an EEG indicating the complete suppression of

electroencephalographic activity. Then, the clips were removed, and the withdrawn blood was reinfused. At the

end of the anesthesia process, the vascular catheters were removed, and the wounds were sutured. The

endotracheal catheter was extubated until there was a recovery of spontaneous respiration and the righting reflex.

Sham-operated rats underwent the same procedures, except for the I/R.

To observe the time-course of I/R-induced changes in the mRNA levels of the apoptosis and

autophagy-related proteins LC3, p53, DRAM and PUMA, the rats (n = 6 in each group) were subjected to a

transient two-vessel occlusion for 10 min and sacrificed at 1.5, 3, 6, 12 and 24 h.

To study the time-course of I/R-induced alterations in the apoptosis-related protein levels of p53, PUMA, Bax,

Bcl-2 and the autophagy-related protein levels of DRAM, LC3 and Beclin 1, the rats (n = 6 in each group) were

subjected to I/R injury and then sacrificed at 1.5, 3, 6, 12 and 24 h.

Sham-operated rats underwent the same procedures, except for the I/R procedure. The left femoral artery was

cannulated to measure the arterial pH, PaCO2, PaO2 and blood glucose concentration. These parameters (Table 1

and Table 2) were measured before and during I/R and 60 min after the I/R insult.

Drug administration

To determine the contributions of propofol to I/R-induced changes in LC3 mRNA and protein levels, the

cellular location of the increased LC3-Ⅱ and the histological characteristics, the rats (n= 6 in each group) were

treated with an intraperitoneal (i.p.) injection of propofol (AstraZeneca UK Limited; 10, 50, and 100 mg/kg) or

intralipid (used as a vehicle control, Sigma, St Louis, MO, USA; 100 mg/kg), an intracerebral ventricle (i.c.v.)

injection of the p53 inhibitor Pifithrin-alpha (PFT-α; Sigma, St Louis, MO, USA; 60 nmol, diluted in 1% DMSO

to a final volume of 5 μl) or the autophagy inhibitor 3-methyladenine (3-MA; Sigma, St Louis, MO, USA; 600

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nmol, diluted in 1% DMSO to a final volume of 5 μl) 10 min after I/R, and the rats were sacrificed 12 h later.

To study the effects of propofol on the I/R-induced changes in p53 mRNA and protein levels, the rats (n = 6

in each group) were treated with an intraperitoneal injection of propofol (10, 50, 100 mg/kg) or vehicle (intralipid,

100 mg/kg), an intracerebral ventricle injection of the p53 inhibitor PFT-α (60 nmol) or the NF-κB inhibitor SN50

(Biomol, Plymouth, PA, USA; 30 μg, diluted in 1% DMSO to a final volume of 5 μl) 10 min after I/R injury, and

the rats were sacrificed 12 h later.

To study the effects of propofol on I/R-induced changes in DRAM and PUMA mRNA levels, alterations in

autophagy-related protein levels of DRAM, Beclin 1, Cathepsin B, Cathepsin D, and apoptosis-related protein

levels of PUMA, Bax and Bcl-2, the rats (n = 6 in each group) were treated with an intraperitoneal injection of

propofol (100 mg/kg) or vehicle (intralipid, 100 mg/kg), an intracerebral ventricle injection of PFT-α (60 nmol) or

SN50 (30 μg) 10 min after I/R injury and sacrificed 12 h later.

Isolation of RNA and real-time quantitative reverse transcription PCR

Total RNA was extracted using the RNAiso Reagent kit (Takara, DaLian, LiaoNing, China). cDNA was

generated by reverse transcription of 2 μg of total RNA using random primers and the PrimescriptTM RT Reagent

Kit (Takara, DaLian, Liaoning, China) in a total reaction volume of 20 μL, according to the manufacturer’s

instructions. The sequences of forward and reverse oligonucleotide primers, specific to the p53, PUMA, DRAM,

LC3 and housekeeping genes, were designed using Primer 5 software. The primers used were the following:

5’-CCC AGG GAG TGC AAA GAG AG-3’and 5’-TCT CGG AAC ATC TCG AAG CG-3’ for p53; 5’-GTG

TGG AGG AGG AGG AGT GG-3’ and 5’-TCG GTG TCG ATG TTG CTC TT-3’ for PUMA; 5’-ATG GCC

ATC TCC GCT GTT TC-3’ and 5’-TGG ATT CCA TTC CAG CTT GGT TA-3’ for DRAM; 5’-CTT CGC CGA

CCG CTG TAA-3’ and 5’-ATC CGT CTT CAT CCT TCT CCT G-3’ for LC3; and 5’-GAC AAT TTT GGC ATC

GTG GA-3’ and 5’-ATG CAG GGA TGA TGT TCT GG-3’ for rat glyceraldehyde-3-phosphate dehydrogenase

(GAPDH).

Real-time quantitative PCR was performed in the iCycler iQ® Multicolor Real-Time PCR Detection System

(Bio-Rad, Hercules, CA, USA). A 20-fold dilution of each cDNA was amplified in a 20 μL volume, using the Fast

Start DNA Master PLUS SYBR Green I (Roche Applied Science, Mannheim, Germany), with 200 nM final

concentrations of each primer. The PCR cycle conditions were 95°C for 10 s and 50 cycles of 95°C for 20 s and

60°C for 30 s. The amplification specificity was evaluated with a melting curve analysis. Threshold cycle Ct,

which correlates inversely with the target mRNA levels, was calculated using the second derivative maximum

algorithm provided by the iCycler software. We used the 2-ΔΔCt method (Livak & Schmittgen, 2001) to calculate

mRNA levels based on Ct values. For each cDNA, the mRNA levels were normalized to GAPDH mRNA levels.

Immunofluorescence analyses

The rats were anesthetized with 4% chloral hydrate and perfused with PBS (pH 7.4) followed by 0.1 mol/L

PBS (pH 7.4) containing 4% paraformaldehyde at 12 h after I/R. Coronal brain sections of 30-μm thickness were

cut with a cryostat. For immunofluorescent labeling, the sections were preincubated for 1 h in 1% normal bovine

serum albumin (BSA) and 0.1% Triton X-100 in PBS and then incubated overnight at 4°C with antibodies against

LC3-Ⅱ (#ab64781; Abcam, Cambridge, MA, USA) in 1% BSA and 0.1% Triton in PBS. The sections were rinsed

three times in PBS and incubated with FITC-conjugated anti-rabbit IgG (#sc-2012; Santa Cruz Biotechnology, CA,

USA ) in a humidified container for 2 h at room temperature. The sections were then rinsed with PBS three times

and incubated with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; #32670; Sigma, St Louis, MO, USA; 5

µg/ml). An LSM 510 Meta confocal microscope was used for confocal laser microscopy. For the double labeling

experiments, the immunoreactive signals were sequentially visualized in the same section with two distinct filters,

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with acquisition performed in separate modes. The sections were viewed under high power (200x) with a

fluorescence microscope (Eclipse TE 2000, Nikon, Japan) that had a Nikon digital camera, and the images were

visualized on a computer monitor. For the quantification of LC3-II immunostaining, 10 microscopic fields (200x

magnification) in each section across ischemic hippocampus regions in the ipsilateral hemisphere were analyzed.

Three sections were used for each animal. The number of cells with LC3-II immunoreactivity in each field was

counted by an examiner who was blind to the experimental conditions.

Histochemical analyses

The rats were anesthetized with 4% chloral hydrate anesthesia (400 mg/kg) and transcardially perfused with

phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS (pH 7.4).

For light microscopy analysis, the brain tissues were quickly removed from the rats and immersed in the same

fixative for 2 hours at 4°C. The samples were processed for paraffin embedding and were cut into 5-µm-thick

sections using an ultramicrotome (Ultracut N; Reichert-Nissei, Tokyo, Japan) and placed on silicone-coated glass

slides. For routine histological studies, the paraffin sections were stained with thionine. Next, the total number of

neurons in the hippocampus CA1 region was statistically analyzed with Stereo Investigator software (MBF

BioScience, Williston, VT, USA). The data of neuronal numbers were converted to a percentage of the control

condition (sham-operated) and expressed as the mean ± standard deviation (SD).

Protein preparation and immunoblotting.

Western blot analyses were performed as described previously (Cui et al., 2012). Hippocampal tissue

samples were homogenized in cold Radio Immunoprecipitation Assay lysis buffer (Beyotime Corporation, Nanjing,

Jiangsu, China) with a 1% protease-inhibitor cocktail (Sigma-Aldrich, USA), followed by centrifugation at

14,000×g for 10 min at 4°C. Protein concentrations were determined using a BCA protein assay kit (Beyotime

Corporation, China). After heating the protein aliquots (30 g) in SDS-PAGE protein loading buffer (Beyotime

Corporation, China) at 95°C for 10 min, we separated them on SDS-PAGE gels and transferred the proteins to

PVDF membranes (Millipore Corporation, Billerica, MA, USA) for immunoblotting. We incubated the

membranes in blocking buffer (5% milk in Tris-buffered saline [TBS] with 0.1% Tween 20) for 1 h at room

temperature, followed by an overnight incubation at 4°C with the following primary antibodies: mouse monoclonal

anti-p53 antibody (#2524; Cell Signaling Technology, Woburn, MA, USA); rabbit polyclonal anti-PUMA

antibody (#4976; Cell Signaling Technology); rabbit polyclonal anti-Bax antibody (#sc-526; Santa Cruz, CA,

USA); rabbit polyclonal anti-Bcl-2 antibody (#2870; Cell Signaling Technology); rabbit polyclonal anti-DRAM

antibody (#905-738-100; Assay Designs & Stressgen Bioreagents, Ann Arbor, MI, USA); rabbit polyclonal

anti-LC3 antibody (#ab62721; Abcam, Cambridge, MA, USA); rabbit polyclonal anti-Beclin 1 antibody (#3738;

Cell Signaling Technology); goat polyclonal anti-Cathepsin B antibody (#sc-6493; Santa Cruz); and goat

polyclonal anti-Cathepsin D antibody (#sc-6486; Santa Cruz). We then washed off the primary antibody three

times in TBST, incubated the membranes with horseradish peroxidase-conjugated secondary antibody (anti-mouse,

anti-rabbit or anti-goat IgG; 1:5000; Santa Cruz) in TBST for 2 h at room temperature and washed them three

times in TBST. Immunoreactivity was detected with enhanced chemoluminescent autoradiography (ECL kit,

Amersham, Arlington Heights, Illinois) and visualized on X-ray film (Kodak, Shanghai, China). GAPDH (#2118;

Cell Signaling Technology) was used as the loading control. The levels of protein expression were quantitatively

analyzed with Sigma Scan Pro 5 and were normalized to a GAPDH loading control.

Statistical Analyses

All data are expressed as the mean ± SD. To establish significance, the data were subjected to a one-way

ANOVA using the GraphPad Prism software statistical package (GraphPad Software, San Diego, CA, USA).

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When a significant group effect was found, post hoc comparisons were performed using the Bonferroni t-test to

examine special group differences. Independent group t-tests were used for comparing two groups. We considered

a result significant when p≤0.05.

Results

Propofol reduces I/R-induced autophagy activation

The results revealed that the mRNA levels of LC3 increased 1.5–24 h following I/R injury, and peak

expression was at 12 h following I/R injury (P<0.01; Fig. 1A). We also examined the protein levels of LC3-I and

LC3-II using Western blot analyses. A remarkable upregulation of the LC3-II ⁄ LC3-I ratio was observed starting

at 3 h following I/R administration (P<0.05), reaching a peak at 12 h after the I/R insult (P<0.01; Fig. 1B and C).

Treatment with propofol (100 mg/kg), PFT-α or 3-MA resulted in a remarkable suppression of the

I/R-induced upregulation of LC3 mRNA levels (P<0.01; Fig. 1D) and LC3 protein levels (P<0.01; Fig. 1E and F)

in the rat hippocampus 12 h following ischemic insult.

In vehicle-treated rats, the number of LC3 II-positive pyramidal neurons in the ischemic pyramidal layers of

the CA1 hippocampus was found to be significantly elevated (P<0.01; Fig. 2A and B). In contrast, propofol (50

and 100 mg/kg), PFT-α and 3-MA dramatically reduced the number of LC3 II-positive neurons (P<0.01; Fig. 2A

and B).

Propofol reduces I/R-induced pyramidal neurons death

An early decrease in the number of hippocampus CA1 pyramidal neurons has been reported after severe

ischemic insults (Cui et al., 2012; Chiara et al., 2006). In the present study, in the vehicle-treated rats, the number

of pyramidal neurons in the ischemic pyramidal layers of the CA1 hippocampus was found to be significantly

reduced 12 h after I/R (P<0.01; Fig. 3B and H). In contrast, propofol (50 and 100 mg/kg), PFT-α and 3-MA

dramatically elevated the number of pyramidal neurons 12 h after I/R (P<0.01; Fig. 3C-H).

There were no remarkable changes in the pH levels, the arterial carbon dioxide (PaCO2) and oxygen (PaO2)

concentrations or blood glucose concentrations before and after the intracerebral ventricular injection of PFT-α or

3-MA or the intraperitoneal injection of propofol in any of the groups (P>0.05; Table 1).

Propofol decreased the upregulation of p53 induced by I/R

Our data revealed that p53 mRNA levels were elevated at 6 and 12 h following the I/R insult (P<0.05 and

P<0.01, respectively; Fig. 4A). A remarkable augmentation in p53 protein levels was observed 12 h following I/R

insult and continued up to 24 h after I/R (P<0.01; Fig. 4B and C). Treatment with the p53 inhibitor PFT-α or the

NF-κB inhibitor SN50 via intracerebral ventricle injection led to a remarkable suppression of the I/R-induced

increases in p53 mRNA (P<0.01; Fig. 4D) and protein (P<0.01; Fig. 4E and F) levels in the rat hippocampus.

Treatment with an intraperitoneal injection of the intravenous anesthetic propofol (100 mg/kg) led to a

remarkable suppression of the I/R-induced increases in p53 mRNA (P<0.01; Fig. 4D) and protein (P<0.01; Fig. 4E

and F) levels in the rat hippocampus at 12 h after the I/R insult.

Activation of the p53-mediated autophagic pathway by I/R.

The p53 target gene damage-regulated autophagy modulator (DRAM) plays important roles in the activation

of autophagy and p53-mediated apoptosis (Crighton et al., 2006). The results demonstrate that DRAM mRNA

levels increased starting 3 h following I/R insult (P<0.05; Fig. 5A). A robust elevation in DRAM protein levels

was observed 6–24 h following I/R insult (P<0.01; Fig. 5B and C). DRAM mRNA levels were dramatically lower

at 12 h following I/R insult in the rat hippocampi treated with PFT-α or SN50 than those treated with vehicle

(P<0.01; Fig. 5D). Likewise, DRAM protein levels were dramatically lower at 12 h after I/R insult in the rat

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hippocampi treated with PFT-α or SN50 than those treated with a vehicle injection (P<0.01; Fig. 5E and F).

The results indicated that Beclin 1 levels increased 6–24 h following I/R insult (P<0.01; Fig. 6A and B).

Beclin 1 protein expression levels were dramatically lower at 12 h after I/R insult in rat hippocampi treated with

PFT-α or SN50 than those treated with vehicle (P<0.01; Fig. 6E and F).

The protein levels of active cathepsin D and cathepsin B were elevated in vehicle-treated rats 12 h after I/R

injury (P<0.01) but were dramatically lower in rats treated with PFT-α or SN50 (P<0.01; Fig. 6C, D, G and H).

The effect of propofol on the expression of autophagy-related proteins after I/R insult

DRAM mRNA levels were remarkably lower 12 h following I/R insult in the rat hippocampi treated with

propofol than those treated with vehicle (P<0.01; Fig. 5D). Likewise, DRAM protein levels were remarkably

lower in the rat hippocampi treated with propofol than those treated with vehicle (Fig. 5E and F).

Beclin 1 protein expression levels were remarkably lower at 12 h following I/R insult in the rat hippocampi

treated with propofol than those treated with vehicle (P<0.01; Fig. 6E and F). Likewise, the protein levels of active

cathepsin D and cathepsin B were also remarkably lower at 12 h after I/R treatment in the rat hippocampi treated

with propofol than those treated with vehicle (P<0.01; Fig. 6C, D, G and H).

Induction of the p53-mediated apoptotic pathway by I/R insult

Likewise, the mRNA levels of the p53 target gene PUMA were elevated 12 to 24 h after I/R administration

(P<0.01 and P<0.05, respectively; Fig. 7A) along with PUMA protein levels during the same time period (P<0.01;

Fig. 7B and C). PUMA mRNA levels were robustly lower at 12 h after I/R treatment in rat hippocampi treated

with intracerebral ventricle injection of the p53 inhibitor PFT-α or the NF-κB inhibitor SN50 than those treated

with intraperitoneal injection of vehicle (P<0.05; Fig. 7D). Likewise, PUMA protein levels were robustly lower in

rat hippocampi treated with intracerebral ventricle injection of PFT-α or SN50 than those treated with vehicle

(P<0.01; Fig. 7E and F).

The levels of Bax and Bcl-2 were also examined with immunoblotting after I/R insult. A significant elevation

in Bax protein levels was observed 6 h after I/R administration (P<0.01; Fig. 8A and B). In contrast, a robust

reduction in Bcl-2 protein levels was observed 6 h after I/R administration (P<0.05; Fig. 8C and D). Bax protein

expression was remarkably lower 12 h after I/R treatment in the rat hippocampi treated with PFT-α or SN50 than

those treated with vehicle (P<0.01; Fig. 8E and F). Bcl-2 protein expression levels were remarkably higher in rat

hippocampi treated with PFT-α or SN50 than those treated with vehicle (P<0.01; Fig. 8G and H).

The effect of propofol on the expression of apoptosis-related proteins after I/R insult

PUMA mRNA levels were remarkably lower at 12 h after I/R treatment in the rat hippocampi treated with

propofol than those treated with vehicle (P<0.05; Fig. 7D). Likewise, PUMA protein levels were remarkably lower

in the rat hippocampi treated with propofol than those treated with vehicle (P<0.01; Fig. 7E and F).

Bax protein expression was remarkably lower at 12 h after I/R treatment in the rat hippocampi treated with

propofol than those treated with vehicle (P<0.01; Fig. 8E and F). Whereas, Bcl-2 protein expression was

remarkably higher in the rat hippocampi treated with propofol than those treated with vehicle at the same time

(P<0.01; Fig. 8G and H).

There were no remarkable changes in the pH levels, the arterial carbon dioxide (PaCO2) and oxygen (PaO2)

concentrations or blood glucose concentrations before and after the intracerebral ventricular injection of PFT-α,

SN50 or the intraperitoneal injection of propofol in any of the groups (P>0.05; Table 2).

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Discussion

Our results indicate that propofol prevents autophagy activation and cell death at clinically relevant

concentrations (Engelhard et al., 2004; Wilson et al., 2002; Tsuchiya, 2002) in models of cerebral ischemia in vivo.

In transient models of global and focal ischemia induced by either MCAO or endothelin-1 intrastriatal injections in

awake rats, propofol has repeatedly been shown to be neuroprotective (Pittman et al., 1997; Wang et al., 2002;

Bayona et al., Gelb et al., 2002). A previous study has reported that propofol can reach the brain as a result of its

high lipid–water coefficient partition and can directly inhibit neuronal cell death in the brain during anesthesia

(Engelhard et al., 2004; Tsuchiya et al., 2002; Gelb et al., 2002; Shyr et al., 1995). Propofol also has been shown to

attenuate reperfusion injury in the rat heart by suppressing autophagic cell death (Noh et al., 2010). These results

demonstrate that the propofol effects on the brain and heart may be significant in the ischemic setting in vivo, as it

may directly suppress autophagy or indirectly modulate the production/concentration of other cytotoxic mediators.

These cytotoxic mediators include free radicals, glutamate, or calcium, which can alter mitochondrial integrity or

trigger autophagy activation (Noh et al., 2010; Javadov et al., 2000; Mattson et al., Klionsky et al., 2000). Our

previous study also suggests that propofol can ameliorate autophagic processes via the reduced expression of

autophagy-related proteins in vitro and in vivo (Cui et al., 2012). This suppression improves cell survival, which

affords a novel explanation for the advantageous pleiotropic effects of propofol in the nervous system.

In our previous studies, we found that cerebral ischemia-reperfusion resulted in autophagy activation.

Blocking autophagy partially inhibited cerebral-ischemic injury, suggesting that autophagy may contribute to the

ischemic injury of hippocampal neurons (Cui et al., 2012; Qin et al., 2010). Therefore, therapeutic strategies to

suppress autophagy-induced neuronal death may demonstrate beneficial effects in the treatment of brain injury

(Koike et al., 2008). In this study, we observed that I/R-induced loss of hippocampal neurons and the expression of

autolysosome-related proteins were both robustly inhibited by propofol at a dose of 100 mg/kg. Therefore, our

results indicate that propofol prevents autophagic cell death in cerebral ischemia models in vivo.

Autophagic and apoptotic cell death are two forms of programmed cell death that play crucial roles in the

removal of unneeded and abnormal cells (Yan et al., 2006; Yan et al., 2007). While these two forms of

programmed cell death are morphologically distinct, recent studies demonstrate that autophagic and apoptotic cell

death utilize some common regulatory mechanisms (Rubinsztein et al., 2005). One study showed that p53

participates in excitotoxic neuronal death, most likely through both apoptotic and autophagic mechanisms (Wang

et al., 2009). However, it is not clear whether p53 also mediates the signaling pathway for autophagy and apoptosis

during and after I/R-induced brain damage. In the current study, we assessed the downstream mechanisms of p53

in mediating cerebral I/R injury. Our results demonstrated that during the process of neuronal cell death induced

by I/R, upregulation of p53 and DRAM, Beclin 1, active cathepsin D, active cathepsin B and LC3-II, all proteins

involved in autophagy, was observed. In addition, the current study demonstrates that I/R induced the expression

of the pro-apoptotic p53 target genes PUMA and Bax and the downregulation of the anti-apoptotic protein Bcl-2,

all of which are proteins involved in apoptosis.

The present study examined the effects of the p53-specific inhibitor PFT-α on I/R-induced autophagy

activation to evaluate whether p53 mediates the signaling pathway for autophagy in cerebral ischemia. The

chemical compound PFT-α is a small molecule inhibitor of p53 transcription activity. The present results indicated

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that PFT-α suppressed I/R-induced autophagy activation. These results indicate that autophagy activation may be

dependent, at least partially, on a p53 mechanism. LC3-Ⅱis required for the formation of autophagosomes and has

been defined as a biomarker of autophagosomes in mammalian cells (Kabeya et al., 2000). LC3-Ⅱis the cleaved

and lapidated form of the cytosolic LC3-Ⅰ.The post-translation modification allows LC3 to translocate to

autophagosomal membranes. The present results showed that p53-specific inhibitor PFT-α inhibited I/R-induced

elevations of LC3-Ⅱ. These data suggest that upregulation of LC3-Ⅱappears to depend, at least partially, on a p53

mechanism.

p53 is an important modulator of cell death and survival, and its inhibition could be a therapeutic approach to

several neuropathologies (Green et al., 2006). We also examined the effects of the p53-specific inhibitor PFT-α on

I/R-triggered hippocampal cell death to evaluate the contribution of p53-mediated pathways to I/R-induced

neuronal death. The results indicated that PFT-α markedly decreased I/R-induced hippocampal damage. 3-MA is a

relatively selective inhibitor of the class III phosphatidylinositol kinase, which is the mammalian homolog of yeast

vps34. Vsp34 is a vacuole sorting protein required for autophagy and for protein sorting from the Golgi to the

vacuole or lysosome (Schu et al., 1993). 3-MA strongly suppresses the maturation of autophagosomes and has

been commonly used for dissecting the role of autophagy in cellular functions (Wang et al., 2008; Qin et al., 2003).

The present study indicated that 3-MA significantly decreased I/R-induced hippocampal neuron death. Blockage

of autophagy by 3-MA partially inhibited cerebral-ischemic injury, also suggesting that autophagy contributes to

the ischemic injury of hippocampal neurons. Similar to the p53-specific inhibitor PFT-α and the autophagy-

specific inhibitor 3-MA, the present study also demonstrated that propofol reversed the I/R-induced upregulation

of p53 and significantly reduced the I/R-induced expression of LC3-Ⅱ and autophagic damage in the hippocampus.

The results indicate that propofol prevented autophagic cell death, likely by inhibiting p53 in the hippocampus

after I/R insult.

DRAM is a p53 target gene that encodes a lysosomal protein. DRAM is indispensable for p53-mediated

apoptosis and is a new regulator of p53-induced autophagy (Crighton et al., 2006). Various stimuli induce

autophagy in mammalian cells; however, little is known regarding the regulatory pathways downstream of these

stimuli. The discovery of DRAM as a novel regulator of autophagy links p53 to both apoptosis and autophagy

(Crighton et al., 2006). In the present study, we found that the upregulation of DRAM and other autophagy

regulators could be induced by I/R. The NF- κB inhibitor SN50 and the p53-specific inhibitor PFT-α suppressed

the elevations in DRAM, Beclin 1, LC3-II, active cathepsin D and cathepsin B induced by I/R. This finding

demonstrates that NF-κB/p53 activates autophagy through DRAM in response to I/R-triggered cell death. Similar

to SN50 and PFT-α, propofol also reversed I/R-induced upregulation of DRAM, thus reversing I/R-induced

elevations of LC3-II, Beclin 1, active cathepsin D and cathepsin B protein levels in the hippocampus. These results

indicate that propofol prevented autophagic cell death, likely through the DRAM-mediated NF-κB/p53 signaling

pathway.

Autophagic cell death can also regulate the apoptotic cascade either positively or negatively (Martin et al.,

2004). In many conditions, autophagy and apoptosis can occur in the same cell in a sequential or concurrent

fashion (Akdemir et al., 2006; Gajewska et al., 2005). A sophisticated cross-talk also exists between autophagy and

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apoptosis. Our previous study indicated that apoptosis was accompanied by an early and significant alteration of

autophagosomal and lysosomal components, as examined by electron microscopy, immunoblotting and

immunofluorescence (Cui et al., 2012). These results demonstrate a possible role of autophagy in I/R-induced

neuronal injury. Recent studies suggest that autophagy has a remarkable impact on the propagation of apoptotic

signals (Wang et al., 2009). Our studies also indicate that the autophagic signaling pathway has a remarkable

impact on the propagation of apoptotic signals induced by I/R. Inhibition of autophagy activation also attenuated

apoptotic cell death. Cathepsins may be associated with the molecular switch between apoptosis and autophagy

during cross-talk between different cell death programs. Several lines of evidence support a positive role for

autophagy and the lysosomal system in apoptosis (Wang et al., 2009; Yan et al., 2007; Uchiyama et al., 2001;

Bampton et al., 2005; Yan et al., 2006). Recent studies have found that certain cathepsins can directly or indirectly

activate caspases. The latter is mediated through cleavage of BID by active cathepsin B. The BID cleavage leads to

mitochondrial translocation and cytochrome C release (Reiners et al., 2002). The present study also showed that

the increases in active cathepsin B induced by I/R accompanied apoptosis activation. Inhibiting the expression of

active cathepsin B by propofol, PFT-α or SN50 could partly prevent apoptosis activation.

In most cases, p53-induced apoptosis activation depends on the expression of the p53 target protein, PUMA

(Jeffers et al., 2003). Recent studies demonstrate that PUMA is a dominant regulator of oxidative stress-induced

Bax activation and neuronal apoptosis (Steckley et al., 2007). Bcl-2 is a distinguished anti-apoptotic protein that

binds to Bax to inhibit the formation of a mitochondrial permeability transition pore (Mahajan et al., 1998). It has

also been reported that Bcl-2 forms a complex with Beclin 1 (Gayta´n et al., 2008). Therefore, it is now believed

that Bcl-2 inhibits both apoptosis and autophagy (Saeki et al., 2000). p53-mediated downregulation of Bcl-2 and

upregulation of Bax promote cytochrome c release from the mitochondria and caspase-3 activation (Qin et al.,

1999; Chipuk et al., 2004 ; Lahiry et al., 2008). Mitochondria-mediated activation of caspases also plays a major

role in apoptosis (Nijboer et al., 2008; Wyttenbach et al., 2006). Activation of the apoptotic cascade including the

release of mitochondria cytochrome c and caspase-1 and -3 activation has been found in excitotoxin-induced cell

death in addition to the induction of p53, PUMA and Bax, and downregulation of Bcl-2 (Qin et al., 1999; Cao et

al., 2005; Liang et al., 2005). To identify whether p53 regulates the signaling pathway for apoptosis in cerebral

ischemia, the current study assessed the effects of the p53-specific inhibitor PFT-α or the NF- κB inhibitor SN50

on I/R-induced apoptosis activation. In our study, the increase in PUMA and Bax and the decrease in Bcl-2

induced by I/R were inhibited by PFT-α and SN50, indicating that NF-κB/p53 activates apoptosis through PUMA

in response to I/R-triggered cell death. Similar to SN50 and PFT-α, propofol also reversed the I/R-induced

upregulation of PUMA, thus reversing the I/R-induced elevations of Bax and decreasing Bcl-2 protein levels in the

hippocampus. These results indicate that propofol prevents apoptotic cell death, likely through a PUMA-mediated

NF-κB/p53 signaling pathway.

There are a number of issues in this study that still must be clarified. (1) There are only a limited number of

reliable biomarkers available for detecting autophagy in mammalian cells. It is generally agreed that LC3 is a

cellular marker for autophagy activation. LC3-Ⅱis required for the formation of autophagosomes and has been

defined as a biomarker of autophagosomes in mammalian cells (Kabeya et al., 2000). In the present study, the ratio

of LC3-Ⅱ/LC3-Ⅰsignificantly increased after I/R treatment. However, electron microscopic examination of the

formation of autophagosomes is the most reliable method for detection of autophagy activation. The current study

does not provide morphological evidence showing autophagy activation induced by cerebral I/R injury in vivo.This

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is the first limitation of our methodology. (2) For routine histological studies, the paraffin sections of rat brain

were stained with thionine. However, the current study does not provide histological investigation such as TUNEL

staining to show apoptosis induced by cerebral I/R injury in vivo.This is the second limitation of our

methodology.

Conclusions

Our results demonstrate that the NF-κB/p53 pathway has a remarkable impact on the propagation of

autophagic and apoptotic cell death signals following cerebral ischemic exposure. Accordingly, studying the role

of the NF-κB-dependent p53 signal transduction pathway may provide a novel therapeutic option for cerebral

ischemic diseases. Our results also indicate that propofol attenuated cell death by inhibiting the expression of p53

and the p53 target genes associated with the autophagy and apoptosis in the rat hippocampus after cerebral I/R

insult.

Acknowledgements

The work was supported by grants from the Natural Science Foundation of Shanghai Jiaotong University

(2012043), the Ph.D. Programs Foundation of the Ministry of Education of China (20120073110087) and the Ph.D.

Innovation Fund Project to Dr. Cui from Shanghai Jiaotong University (BXJ201237). The funders had no role in

study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Figure Legends

Figure 1. Propofol decreased LC3-II mRNA and protein levels. To study the time-course of I/R-induced

changes in LC3, the rats were treated with 2-vessel occlusion and sacrificed 1.5, 3, 6, 12 and 24 h after I/R

administration. Hippocampal tissues were dissected for preparation of total RNA for real-time quantitative reverse

transcription PCR (A) or for preparation of hippocampal extracts for immunoblotting (B and C). To study the

effects of propofol, Pifithrin-alpha (PFT-α) and 3-methyladenine (3-MA) on LC3, the rats were administered an

intraperitoneal injection of propofol (10, 50, 100 mg/kg) or vehicle (intralipid, 100 mg/kg) and

intracerebroventricular injection of PFT-α (60 nmol) or 3-MA (600 nmol) 10 min after the onset of ischemia and

were sacrificed 12 h later. Hippocampal tissues were dissected for preparation of total RNA for real-time

quantitative reverse transcription PCR (D) or for preparation of hippocampal extracts for immunoblotting (E and

F). The optical densities of the respective protein bands were analyzed using Sigma Scan Pro 5 and normalized to

the loading control (GAPDH). The data are expressed as the mean ± SD (n = 6). Statistical comparisons were

performed using one-way ANOVA followed by a Bonferroni t-test. * P < 0.05 vs. control group; ** P < 0.01 vs.

control group; # P < 0.05 vs. I/R-treated group; ## P < 0.01 vs. I/R-treated group.

Figure 2. Propofol decreased LC3-II protein expression in the ischemic hippocampus after I/R in rats. (A)

To study the effects of propofol, Pifithrin-alpha (PFT-α) and 3-methyladenine (3-MA) on LC3, the rats were

administered an intraperitoneal injection of propofol (10, 50, 100 mg/kg) or vehicle (intralipid, 100 mg/kg) and an

intracerebroventricular injection of PFT-α (60 nmol) or 3-MA (600 nmol) 10 min after the onset of ischemia and

were sacrificed 12 h later. I/R was induced by 2-vessel occlusion. Images (magnification 200x) were captured

from the same part of the ischemic hippocampus. (B) Quantitative analysis of the number of LC3-II-positive

neurons. The number of LC3-II-positive neurons in the ischemic hippocampus was significantly decreased in the

propofol (50,100 mg/kg), PFT-α and 3-MA -treated rats compared with the vehicle rats. The data are expressed as

a percentage of the sham-operated animals and as the mean±SD; n = 6. Statistical analyses were performed using a

one-way ANOVA followed by a Bonferroni t-test. ** P < 0.01 vs. sham group; ## P < 0.01 vs. I/R-treated group.

Figure 3. Propofol increased the number of the hippocampal pyramidal neurons in the ischemic

hippocampus after I/R in the rats. I/R was induced by 2-vessel occlusion. Vehicle (B) (intralipid, 100 mg/kg) or

Propofol (10, 50, 100 mg/kg) (C-E) was administrated intraperitoneally, and Pifithrin-alpha (PFT-α; 60 nmol) (F)

or 3-methyladenine (3-MA; 600 nmol) (G) was administered intracerebroventricularly 10 min after the onset of

ischemia. Histochemical morphological analyses were performed 12 h after I/R. (H) Quantitative analysis of the

number of hippocampal pyramidal neurons. The number of hippocampal pyramidal neurons in the ischemic

hippocampus was significantly increased in the propofol, PFT-α and 3-MA-treated rats compared with the

vehicle-treated rats. The data are expressed as the percentage of sham-operated group animals and as the mean

±SD; n= 6. The statistical analysis was performed using a one-way ANOVA. *P < 0.05, **P < 0.01 vs. sham group.

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#P < 0.05, ##P < 0.01 vs. I/R-treated group. Scale bars: lower magnification (I), 50 μm; higher magnification (A-G),

500 μm. so, stratum oriens; sp, stratum pyramidal; sr, stratum radiatum.

Figure 4. The effect of propofol on p53 mRNA and protein levels after I/R insult. To study the time course of

I/R-induced changes in p53, the rats were treated as described in the legend to Fig. 1A-C. Hippocampal tissues

were dissected for the preparation of total RNA for real-time quantitative reverse transcription PCR (A) or for the

preparation of hippocampal extracts for immunoblotting (B and C). To study the effects of propofol,

Pifithrin-alpha (PFT-α) and SN50 on p53, the rats were administered an intraperitoneal injection of propofol (10,

50, 100 mg/kg) or vehicle (intralipid, 100 mg/kg) and an intracerebroventricular injection of PFT-α (60 nmol) or

SN50 (30 μg) 10 min after the onset of ischemia and were sacrificed 12 h later. Hippocampal tissues were

dissected for the preparation of total RNA for real-time PCR (D) or for the preparation of hippocampal extracts for

immunoblotting (E and F). Optical densities of respective protein bands were analyzed with Sigma Scan Pro 5 and

normalized to the loading control (GAPDH). The data are expressed as the mean ± SD; n = 6. Statistical

comparisons were performed using a one-way ANOVA followed by a Bonferroni t-test. * P < 0.05 vs. control

group; ** P < 0.01 vs. control group; ## P < 0.01 vs. I/R-treated group.

Figure 5. The effect of propofol on the mRNA and protein levels of damage-regulated autophagy modulator

(DRAM) after I/R insult. To study the time course of I/R-induced changes in DRAM, the rats were treated as

described in the legend of Fig. 1A-C. The hippocampal tissues were dissected for the preparation of total RNA for

real-time quantitative reverse transcription PCR (A) or for the preparation of hippocampal extracts for

immunoblotting (B and C). To study the effects of propofol, Pifithrin-alpha (PFT-α) and SN50 on DRAM

induction, the rats were administered an intraperitoneal injection of propofol (100 mg/kg) or vehicle (intralipid,

100 mg/kg) and an intracerebroventricular injection of PFT-α (60 nmol) or SN50 (30 μg) 10 min after the onset of

ischemia and were sacrificed 12 h later. The hippocampal tissues were dissected for the preparation of total RNA

for real-time quantitative reverse transcription PCR (D) or for preparation of hippocampal extracts for

immunoblotting (E and F). The optical densities of respective protein bands were analyzed with Sigma Scan Pro 5

and normalized to loading control (GAPDH). The data are expressed as the mean ± SD; n = 6. Statistical

comparisons were performed with one-way ANOVA followed by a Bonferroni t-test. * P < 0.05 vs. sham group; **

P < 0.01 vs. sham group; ## P < 0.01 vs. I/R-treated group.

Figure 6. The effect of propofol on the expression of autophagy-related proteins after I/R insult. To study the

time course of I/R-induced changes in Beclin 1, the rats were treated as described in the legend to Fig. 1B and C.

Hippocampal tissues were dissected for preparation of hippocampal extracts for immunoblotting (A and B). To

study the effects of propofol (100 mg/kg), Pifithrin-alpha (PFT-α; 60 nmol) and SN50 (30 μg) on Beclin 1,

cathepsin D and cathepsin B, the rats were treated as described in the legend to Fig. 5E and F. Hippocampal tissues

were dissected for preparation of total lysates for immunoblotting (C-H). The optical densities of the respective

protein bands were analyzed with Sigma Scan Pro 5. The data are expressed as the mean ± SD; n = 6. Statistical

comparisons were performed using a one-way ANOVA followed by a Bonferroni t-test. ** P < 0.01 vs. sham group;

## P < 0.01 vs. I/R-treated group.

Figure 7. The effect of propofol on the mRNA and protein levels of p53-upregulated modulator of apoptosis

(PUMA) after I/R insult. To study the time course of I/R-induced changes in PUMA, the rats were treated as

described in the legend of Fig. 1A-C. Hippocampal tissues were dissected for the preparation of total RNA for

real-time quantitative reverse transcription PCR (A) or for the preparation of hippocampal extracts for

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

immunoblotting (B and C). To study the effects of propofol (100 mg/kg), Pifithrin-alpha (PFT-α; 60 nmol) or

SN50 (30 μg) on PUMA induction, the rats were treated as described in the legend to Fig. 5D- F. Hippocampal

tissues were dissected for preparation of total RNA for real-time quantitative reverse transcription PCR (D) or for

preparation of hippocampal extracts for immunoblotting (E and F). The optical densities of respective protein

bands were analyzed with Sigma Scan Pro 5 and normalized to the loading control (GAPDH). The data are

expressed as the mean ± SD; n = 6. Statistical comparisons were performed using a one-way ANOVA followed by

a Bonferroni t-test. * P < 0.05 vs. control group; ** P < 0.01 vs. control group; # P < 0.05 vs. I/R-treated group; ## P

< 0.01 vs. I/R-treated group.

Figure 8. The effect of propofol on the expression of apoptosis-related proteins after I/R insult. To study the

time course of I/R-induced changes in Bax, the rats were treated as described in the legend of Fig. 1B and C.

Hippocampal tissues were dissected for preparation of hippocampal extracts for immunoblotting (A and B). To

study the effects of propofol (100 mg/kg), Pifithrin-alpha (PFT-α; 60 nmol) or SN50 (30 μg) on Bax induction, the

rats were treated as described in the legend to Fig. 5 E- F. Hippocampal tissues were dissected for the preparation

of hippocampal extracts for immunoblotting (E and F). To study the time course of I/R-induced changes in Bcl-2,

the rats were treated as described in the legend to Fig. 1B and C. Hippocampal tissues were dissected for

preparation of hippocampal extracts for immunoblotting (C and D). To study the effects of propofol (100 mg/kg),

PFT-α (60 nmol) or SN50 (30 μg) on Bcl-2 induction, the rats were treated as described in the legend to Fig. 5E- F.

Hippocampal tissues were dissected for the preparation of hippocampal extracts for immunoblotting (G and H).

The optical densities of the respective protein bands were analyzed with Sigma Scan Pro 5 and normalized to the

loading control (GAPDH). The data are expressed as the mean ± SD; n = 6. Statistical comparisons were

performed using a one-way ANOVA followed by a Bonferroni t-test. * P < 0.05 vs. control group; ** P < 0.01 vs.

control group; # P < 0.05 vs. I/R-treated group; ## P < 0.01 vs. I/R-treated group.

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Table 1. Physiological parameters.

Groups Time MAP(mmHg) PH PaCo2(mmHg) Po2(mmHg) GI(mg/dl)

Sham Baseline 106±6 7.35±0.02 35.3±2.6 145.3±18.2 166±25

Ischemia 102±5 7.30±0.03 37.8±2.7 142.2±16.5 156±18

Recovery 105±5 7.42±0.03 39.5±2.3 139.5±15.1 159±23

I/R+Int(100 mg/kg) Baseline 105±6 7.39±0.01 40.2±3.1 143.5±15.6 172±21

Ischemia 41±3a 7.42±0.03 39.8±2.6 143.6±18.8 168±25

Recovery 116±4 7.41±0.03 38.8±2.3 140.6±16.5 163±23

I/R+Prop(10 mg/kg) Baseline 105±8 7.39±0.03 37.7±2.5 142.2±16.5 165±22

Ischemia 41±2a 7.41±0.02 38.4±2.3 143.3±18.6 161±19

Recovery 113±6 7.43±0.03 39.3±2.2 141.3±16.1 159±18

I/R+Prop(50 mg/kg) Baseline 108±3 7.38±0.02 38.5±3.2 143.2±18.2 158±17

Ischemia 38±3a 7.41±0.01 39.3±3.5 143.3±16.2 162±16

Recovery 113±5 7.42±0.03 40.3±2.2 143.1±11.3 163±19

I/R+Prop(100 mg/kg) Baseline 105±6 7.39±0.02 39.3±2.6 141.3±18.2 165±15

Ischemia 39±2a 7.38±0.03 40.5±2.9 142.2±16.6 165±17

Recovery 112±5 7.42±0.01 39.6±2.3 144.1±12.9 169±18

I/R+PFT-α(60 nmol) Baseline 105±7 7.39±0.02 39.8±2.8 142.3±16.8 163±18

Ischemia 40±2 a 7.43±0.03 38.6±2.5 144.6±15.9 168±19

Recovery 115±5 7.39±0.02 36.8±2.7 139.5±16.9 169±21

I/R+3-MA(600 nM) Baseline 106±6 7.37±0.02 38.3±3.1 138.6±15.5 165±18

Ischemia 39±2a 7.43±0.03 38.6±2.3 143.3±16.8 155±19

Recovery 115±3 7.39±0.02 38.9±3.1 140.3±18.3 166±23

All values are the mean±SD. Arterial blood gas tensions include PaO2, PaCO2, PH and GI.

MAP, mean arterial pressure; PaO2, arterial oxygen pressure; PaCO2, arterial carbon dioxide pressure; GI, glucose.

a Controlled parameter.

1

Table 2. Physiological parameters.

Groups Time MAP(mmHg) PH PaCo2(mmHg) Po2(mmHg) GI(mg/dl)

Sham Baseline 105±7 7.33±0.02 37.3±2.6 145.3±18.5 165±16

Ischemia 103±6 7.32±0.03 38.7±2.7 141.2±16.3 158±19

Recovery 109±6 7.40±0.03 39.5±2.5 138.5±15.6 160±22

I/R+ Int (100 mg/kg) Baseline 105±5 7.39±0.02 39.5±3.2 142.2±18.5 159±15

Ischemia 39±2a 7.40±0.01 38.3±3.5 142.3±16.1 165±19

Recovery 113±6 7.41±0.03 39.3±2.2 140.1±11.5 162±16

I/R+Prop(100 mg/kg) Baseline 108±7 7.38±0.02 40.3±2.6 142.3±18.3 163±18

Ischemia 38±3a 7.39±0.03 39.5±2.9 140.2±16.5 163±19

Recovery 113±3 7.41±0.01 38.6±2.3 143.1±13.9 166±16

I/R+PFT-α(60 nmol) Baseline 106±8 7.38±0.02 39.5±2.8 140.3±15.8 165±19

Ischemia 39±2 a 7.42±0.03 39.6±2.5 143.6±14.9 169±16

Recovery 115±8 7.38±0.02 37.8±2.7 140.5±15.9 168±22

I/R+SN50(30 μg) Baseline 106±6 7.39±0.02 37.3±3.1 139.6±14.5 166±19

Ischemia 40±3a 7.42±0.03 39.6±2.5 141.3±18.8 158±16

Recovery 113±5 7.38±0.02 39.9±3.2 142.3±17.3 162±22

All values are the mean±SD. Arterial blood gas tensions include PaO2, PaCO2, PH and GI.

MAP, mean arterial pressure; PaO2, arterial oxygen pressure; PaCO2, arterial carbon dioxide pressure; GI, glucose.

a Controlled parameter.

Highlights

Propofol prevents autophagy activation induced by cerebral ischemia-reperfusion insult.

Propofol prevents pyramidal neurons death induced by cerebral ischemia-reperfusion insult.

Propofol reduces the upregulation of p53 induced by cerebral ischemia-reperfusion insult.

propofol inhibits autophagy and apoptosis activation through NF-κB/p53 signaling pathway.