ORIGINAL ARTICLE

Expression of HIF-1a and MDR1/P-glycoprotein in refractorymesial temporal lobe epilepsy patients and pharmacoresistanttemporal lobe epilepsy rat model kindled by coriaria lactone

Yaohua Li • Jianbin Chen • Tianfang Zeng •

Ding Lei • Lei Chen • Dong Zhou

Received: 2 December 2013 / Accepted: 11 February 2014

� Springer-Verlag Italia 2014

Abstract Hypoxia-inducible factor-1a (HIF-1a) is

thought to mediate pharmacoresistance in tumor by inducing

Pgp overexpression. We aimed to investigate the expression

of HIF-1a and MDR1/P-glycoprotein in refractory epilepsy,

to explore the correlation of HIF-1a with epilepsy multidrug

resistance. We collected hippocampus and mesial temporal

lobe (MTL) cortex of refractory mesial temporal lobe epi-

lepsy (mTLE) patients that underwent surgery, and estab-

lished a pharmacoresistant TLE rat model kindled by coriaria

lactone. We used real-time quantitative PCR (RQ-PCR) and

western blot to investigate expression of HIF-1a and MDR1

in hippocampus and MTL/entorhinal cortex. We found that

the expression of HIF-1a and MDR1, at both mRNA and

protein levels, were up-regulated in hippocampus and MTL

cortex of mTLE patients compared with the control cortex

(all P \ 0.05), and increased in hippocampus and entorhinal

cortex of kindled rat model versus the control group (all

P \ 0.05). These results demonstrated the overexpression of

HIF-1a and MDR1/Pgp in hippocampus and MTL/entorhi-

nal cortex of mTLE patients and the pharmacoresistant TLE

rat model. HIF-1a may have a regulatory effect on MDR1

expression in refractory epilepsy, which is probably con-

sistent with MDR mechanism in tumor.

Keywords HIF-1a � Pgp � Multidrug resistance � mTLE �Epilepsy rat model � Coriaria lactone

Introduction

Resistance to multiple anti-epileptic drugs (AEDs) has

been an important clinical challenge in refractory epilepsy

therapy for neurologists. Numerous studies have found that

overexpression of multidrug transporters, such as the

multidrug resistance gene 1 (MDR1) product P-glycopro-

tein (Pgp), in the blood–brain barrier (BBB) restricted

anticonvulsant effect by promoting AEDs efflux [1–4].

Numerous studies have revealed that overexpression of

MDR1/Pgp conferred multidrug resistance in cancer [5, 6],

and hypoxia-inducible factor-1 (HIF-1) may be a primary

factor regulating the expression of MDR1/Pgp [7, 8]. HIF-1

consists of a heterodimer of HIF-1a and HIF-1b, in which

HIF-1a determines the biological activity associated with

hypoxic adaptation and pathological response [9]. A study

revealed that the MDR1 gene-promoter contains a func-

tional HIF-1a binding site known as classical hypoxia

response element (HRE) [10]. Recurrent seizures and fre-

quent epileptic discharges may also cause ambient hypoxia,

resulting in HIF-1a accumulation to adapt to hypoxic

environments.

Considering those evidences, we hypothesized that HIF-

1a has a regulatory function in MDR1 expression in

refractory epilepsy resembling in tumors. Temporal lobe

epilepsy (TLE) accounts for the largest proportion of

refractory epilepsy, and hippocampal sclerosis (HS) is the

most frequent pathological finding in TLE [11]. Therefore,

Lei Chen and Dong Zhou, as the co-corresponding authors,

contributed equally to this study.

Y. Li � T. Zeng � L. Chen (&) � D. Zhou (&)

Department of Neurology, West China Hospital, Sichuan

University, No. 37 Wainan Guoxue Road,

Chengdu 610041, Sichuan, China

e-mail: leilei_25@126.com

D. Zhou

e-mail: zhoudong66@yahoo.de

J. Chen � D. Lei

Department of Neurosurgery, West China Hospital, Sichuan

University, No. 37 Wainan Guoxue Road,

Chengdu 610041, Sichuan, China

123

Neurol Sci

DOI 10.1007/s10072-014-1681-0

we designed this study to investigate the expressions of

HIF-1a and MDR1 in refractory mesial temporal lobe

epilepsy (mTLE) and in a pharmacoresistant TLE Spra-

gue–Dawley (SD) rat model kindled by coriaria lactone

(CL), using real-time quantitative PCR (RQ-PCR), and

western blot (WB) for analysis.

Materials and methods

mTLE patients and control group

We planned to collect the hippocampus and MTL cortex of

mTLE patients. Patients recruited in this study were diag-

nosed with refractory epilepsy by neurologists according to

the definition of pharmacoresistant epilepsy [12]. The 24-h

EEG monitoring indicated that epilepsy-like waves origi-

nated from unilateral temporal lobe. In addition, there was

ipsilateral hippocampal sclerosis identified by MRI or low

metabolism in ipsilateral mesial temporal lobe identified by

PET, without other pathological changes. Surgery was

determined with neurologist and neurosurgery specialist

consultation. In operation, cortical electrode monitoring

confirmed that epilepsy-like waves originated from the

interior temporal lobe, thus anterior temporal lobe resection

was performed. HS was confirmed by frozen pathological

examination. It was infeasible to obtain brain tissues of

normal hippocampus and mesial temporal lobe (MTL) cor-

tex, so that normal temporal cortex tissues were used as

negative control. All patients in control group did not have

history of epilepsy and other systematic diseases.

The study was approved by the Ethics Committee of

West China Hospital. Informed consents were obtained

from the patients and their legal guardians on the use of

their brain tissues in this research. Finally, we collected

five refractory mTLE patients with the hippocampus and

MTL cortex and five cases with normal temporal lobe

cortex as control group. The brain tissues were separated as

needed and immediately preserved in liquid nitrogen. The

clinical data are shown in Table 1.

Pharmacoresistant TLE rat model

The lack of normal hippocampus as control compromised

the validity of study in patients. As supplement, the study

was also performed in a refractory epilepsy rat model. In

our previous study, a kindled Sprague–Dawley (SD) rat

model induced by CL was confirmed as a refractory TLE

model [13]. This epilepsy model is similar to human mTLE

with MTL epileptic genesis and pharmacoresistant prop-

erties [13]. We also have received a Chinese patent for this

epilepsy animal model.

The animal study was approved by the Experimental

Animal Management Institute of Sichuan University and

strictly performed in compliance with the ‘‘Laboratory

Animal Welfare Protection Law of China’’. Fifteen healthy

male SD rats aged 6–8 weeks and weighing 100–120 g were

used. The rats were acclimated under laboratory conditions

Table 1 Clinical data of mTLE patients group and control group

(a) Clinical data of mTLE patients group

Case Gender Age, y Seizure type Duration, y EEG, sp ori MRI/PET

1 M 20 CPS, SGS 6 L-T L-HS/–

2 M 20 CPS, SGS 11 L-T L-HS/–

3 M 19 CPS, SGS 8 L-T L-HS/–

4 F 36 CPS, SGS 15 R-T R-HS/–

5 M 24 CPS, SGS 9 R-T N/R-Ta

(b) Clinical data of control group

Case Gender Age, y Tissue sources

1 M 49 Operative route of benign neoplasm in deep area of brain

2 F 29 Adjacent normal cortex in surgical evacuation of IH

3 F 47 Adjacent normal cortex in surgical evacuation of IH

4 M 36 Adjacent normal cortex in surgical evacuation of IH

5 M 39 Operative route of benign neoplasm in deep area of brain

mTLE mesial temporal lobe epilepsy, M male, F female, y year, CPS complex partial seizure, SGS secondarily generalized seizure, sp ori spikes

origin, L left, R right, T temporal lobe, HS hippocampal sclerosis, N normal, IH intracerebral hematoma

– indicates no PETa Indicates PET show low metabolism

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123

for 1 week before the start of the experiment. CL is an epi-

leptogenic agent extracted from a traditional Chinese herb

coriaria [14], containing tutin (C15H19O6) and coriamyrtin

(C15H18O5) at a concentration of 5 mg/ml (tutin[50 %).

SD rats were randomly divided to experimental (n = 10)

and control (n = 5) groups. The rats in experimental group

were intramuscularly injected with CL 0.4 ml/kg every 72 h,

while in control group with same dose normal sodium. Sei-

zures were graded according to Racine’s five-stage scale

(1972) [15]. The rats were considered as completely kindled

if they had five or more consecutive stage 4 or 5 seizures with

generalized high-amplitude epileptiform discharges on EEG

[13]. After a maximum of 18 times of CL injections, five rats

in experimental group were successfully kindled. In control

group there was no unexpected deaths. Brain tissues of the

kindled group and control group were removed immediately

after deep anesthesia with 6 % chloral hydrate by peritoneal

injection. The hippocampus and entorhinal cortex were

quickly dissected out and preserved in liquid nitrogen.

RQ-PCR

Total RNA was extracted according to Trizol method

(Invitrogen, USA). RNA integrity was analyzed by 1 %

agarose gel electrophoresis. The b-actin was taken as an

internal control. PCR primers and TaqMan probe were

designed and synthesized by Shanghai Shenggong (China).

The sequences are shown in Table 2. RQ-PCR was per-

formed with the FTC2000 PCR system (Funglyn, Canada).

The following conditions were used for amplification:

94 �C for 2 min; 94 �C for 20 s; 54 �C (MDR1, b-actin)/

50 �C (HIF-1a) for 20 s; and 60 �C for 30 s for 45 cycles.

PCR-amplified products qualitatively detected by 2.0 %

agarose gel electrophoresis. The expression rate between

groups was calculated according to the equation derived by

Livak and Schmittgen [16]: expression rate (R) = 2-DDCT.

CT denotes the number of cycles at which the fluorescent

signals in the reaction system are detected by the thermal

cycler, DCT = CT (target gene) - CT (b-actin), and

DDCT = DCT (trial group) - DCT (control group).

WB

Tissues samples (100 mg) were homogenized and centri-

fuged. The protein concentration was determined by BCA

protein assay kit (Pierce, USA) and adjusted to 2 lg/ll. Each

protein sample (10 ll) was resolved by SDS-PAGE (Amer-

sham 80-6418-77, USA) and wet transferred to PVDF mem-

brane (Amersham TE22, USA). The samples were blocked

and then incubated with the primary antibody diluted at

1:1,000 (mouse anti-HIF-1a monoclonal antibody, Novus,

USA; rabbit anti-MDR1 polyclonal antibody, Boaosen, China)

at 4 �C overnight. The membranes were washed and then

incubated with HRP-labeled goat anti-mouse (for HIF-1a) or

anti-rabbit (for MDR1) secondary antibody (1:10,000, Pierce,

USA) at room temperature for 1 h, separately. The bands were

detected using a chemiluminescent substrate (Millipore,

USA). Theb-actin was used as an internal control. Gray values

were measured using ImageJ Analysis Software (NIH). The

relative expressions of samples in different duplications were

standardized by a same sample of control group.

Statistical analysis

Data were processed by SPSS16.0 and expressed as

mean ± standard deviation (SD). The results were com-

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

by least-significant difference (LSD) test for multiple

intergroup comparisons as needed. All tests were two

sided, and P \ 0.05 was considered statistically significant.

Results

RQ-PCR

In the human brain tissue samples, the R value of HIF-1amRNA was 2.91 in the hippocampus and 3.39 in the MTL

cortex versus the control cortex, with significant differ-

ences, respectively (both P \ 0.05). The R value of MDR1

mRNA in the hippocampus and MTL cortex were 2.71 and

2.87 versus the normal cortex, respectively (both

P \ 0.05). No significant difference of HIF-1a mRNA, as

well as MDR1 mRNA, was obtained between hippocampus

and MTL cortex (both P [ 0.05). See in Fig. 1.

Table 2 Sequences of RQ-PCR primers and probes for HIF-1a,

MDR1, and b-actin

Gene Sequences

HIF-1a

Primer

Forward 50-TGCTGATTTGTGAACCCATT-30

Reverse 50-CCAAAGCATGATAATATTCAT-30

TaqMan probe 50-CTCAGTCGACACAGCCTC-30

MDR1

Primer

Forward 50-GCCGAAAACATTCGCTATG-30

Reverse 50-TCTCACCAACCAGGGTGT-30

TaqMan probe 50-CTGTCAAGGAAGCCAATGCC-30

b-Actin

Primer

Forward 50-AAGGCCAACCGCGAGAA-30

Reverse 50-CCTCGTAGATGGGCACA-30

TaqMan probe 50-CTGCACCACCAACTGCTTAGC-30

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In the rat samples, the expression rates of HIF-1amRNA in the kindled group were 1.74 in hippocampus

and 1.39 in entorhinal cortex compared with that in the

control group (both P \ 0.05). The mdr1 mRNA

expression rate values were 2.30 and 1.47 in hippocampus

and entorhinal cortex, respectively (both P \ 0.05). See in

Fig. 2.

WB analysis

For the human brain tissue samples, significant differences

were observed in terms of HIF-1a protein expression in the

hippocampus (1.55 ± 0.12) and MTL cortex (1.46 ± 0.06)

versus the control cortex (1.08 ± 0.18) (both P \ 0.05).

Expressions of Pgp in the hippocampus (1.56 ± 0.18) and

Fig. 1 DCT values of HIF-1a(a) and MDR1 (b) mRNA in

human brain tissue samples.

Higher mRNA expression was

indicated by lower DCT.

Asterisk indicates a significant

difference

Fig. 2 DCT values of HIF-1a(a) and mdr1 (b) mRNA in rat

brain samples. Higher mRNA

expression was indicated by

lower DCT. Asterisk indicates a

significant difference

Fig. 3 Relative expression of

HIF-1a protein (a) and Pgp

(b) in human brain samples. The

representative immunoblot

bands are shown below the

histograms. Asterisk indicates a

significant difference

Neurol Sci

123

MTL cortex (1.51 ± 0.12) versus control cortex

(1.08 ± 0.14), both increased significantly (P \ 0.05), which

was consistent with the up-regulation of HIF-1a. There were

no statistical difference between the hippocampus and MTL

cortex for HIF-1a and Pgp expressions. See in Fig. 3.

In the rat brain samples, the expression of HIF-1a in

hippocampus was significantly higher in kindled group

(1.56 ± 0.11) compared with that in control group

(0.97 ± 0.10; P \ 0.05), and in entorhinal cortex was

1.40 ± 0.07 in kindled group versus 1.00 ± 0.11 in control

group (P \ 0.05). Consistent with HIF-1a, in hippocampus

Pgp overexpressed in kindled group (1.75 ± 0.19) com-

pared with control group (0.99 ± 0.12; P \ 0.05), and in

entorhinal cortex kindled group scored at 1.48 ± 0.07

compared with control group scored at 0.99 ± 0.11

(P \ 0.05). See in Fig. 4.

Discussion

Transporter theory commends that transport protein, such as

Pgp, overexpression and over-activity can hinder AEDs

reaching an effective therapeutic concentration in the epi-

leptogenic focus [17]. On the other hand, numerous studies,

in the tumor pharmacoresistance mechanism, have dem-

onstrated that MDR1 expression is modulated by HIF-1a[10, 18]. In the present study, we found that expression of

HIF-1a and MDR1, at mRNA and protein levels, up-regu-

lated in hippocampus and MTL cortex/entorhinal cortex.

The expression of MDR1 in both rat and human are posi-

tively correlated to HIF-1a, at mRNA and notably at protein

levels, indicating the potential relationship between them.

This study was the first, to our knowledge, to report

expression of HIF-1a in refractory epilepsy patients and the

possible relationship between HIF-1a and MDR1. These

findings suggested that HIF-1a may potentially induce

multidrug resistance in epilepsy by up-regulating MDR1,

which was consistent with the MDR mechanism in tumor.

Our study demonstrated that, in mTLE patients and TLE

model, mRNA and protein of HIF-1a up-regulated in

hippocampus and MTL/entorhinal cortex. HIF-1a is

induced in hypoxic condition but rapidly degraded under

normoxic conditions by the ubiquitin–proteasome system

[19]. Recurrent seizures and frequent subclinical electro-

graphic seizure discharges originated from MTL can lead

to enhanced local oxygen consumption and oxygen desat-

urations in whole body [20, 21], resulting in HIF-1aaccumulation especially in MTL. In addition, some studies

have demonstrated that in patients with mTLE, interictal

and periictal hypoperfusion of MTL was found with

SPECT [22, 23], and interictal hypometabolism was

exhibited in MTL with PET [24, 25], which may explain

overexpression and accumulation of HIF-1a in MTL.

Other studies in animals have also demonstrated that

some downstream target genes of HIF-1, such as EPO and

VEGF, participated in epileptic neurogenesis [26, 27]. As

in ischemic/hypoxic encephalopathy studies HIF-1 mani-

fests dual nature of apoptosis and adaptive [28]. Consid-

ering HIF-1a overexpressed in hippocampus, we

speculated that HIF-1a may involve hippocampal neuronal

apoptosis, secondary glial cell proliferation, and incorrect

connection of the neural network. Therefore, it suggested

that HIF-1a may be a core factor involving occurrence and

development of HS.

A limitation of our study is that optimal control brain

tissues of human, i.e., normal hippocampus and normal

MTL cortex, were infeasible to gain. Thus, we adopted a

pharmacoresistant TLE rat model as supplement. More-

over, the correlation of HIF-1a and MDR1/Pgp should be

further detected. In further research, the co-expression of

HIF-1a and Pgp in spatial distribution at cellular and

structural levels based on immunohistochemistry is needed,

and inhibiting HIF-1a expression using RNA-interference

technology in the rat model will be studied to verify the

possible regulation effect of HIF-1a on MDR1 in refractory

epilepsy.

Fig. 4 Relative expression of

HIF-1a protein (a) and Pgp

(b) in rat brain samples. The

representative immunoblot

bands are shown. Asterisk

indicates a significant difference

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In summary, both HIF-1a and MDR1 up-regulated in

the hippocampus and MTL/entorhinal cortex in mTLE

patients and the pharmacoresistant TLE rat model. HIF-1amay have a regulatory effect on MDR1 expression in

pharmacoresistant epilepsy, which is similar to the multi-

drug resistance mechanism in tumor. The present research

is likely to provide new insights on pharmacoresistance

mechanism in refractory epilepsy.

Acknowledgments This work was supported by the National Nat-

ural Science Foundation of China (no. 81371425), the scientific

research foundation of Sichuan University for outstanding young

scholars (no. 2082604164246), Sichuan Province basic research plan

project (no. 2013JY0168) and Chengdu City Science and Technology

Bureau fund (no. 12DXYB209JH-002).

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