Neurol Sci DOI 10.1007/s10072-014-1681-0
ORIGINAL ARTICLE
Expression of HIF-1a and MDR1/P-glycoprotein in refractory mesial temporal lobe epilepsy patients and pharmacoresistant temporal 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 Ó 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 epilepsy (mTLE) patients that underwent surgery, and established 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/entorhinal cortex of mTLE patients and the pharmacoresistant TLE rat model. HIF-1a may have a regulatory effect on MDR1 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: [email protected] D. Zhou e-mail: [email protected] J. Chen D. Lei Department of Neurosurgery, West China Hospital, Sichuan University, No. 37 Wainan Guoxue Road, Chengdu 610041, Sichuan, China
expression in refractory epilepsy, which is probably consistent 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-glycoprotein (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 functional HIF-1a binding site known as classical hypoxia response element (HRE) [10]. Recurrent seizures and frequent epileptic discharges may also cause ambient hypoxia, resulting in HIF-1a accumulation to adapt to hypoxic environments. Considering those evidences, we hypothesized that HIF1a 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,
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Neurol Sci 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 2
M
49
Operative route of benign neoplasm in deep area of brain
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 PET a
Indicates PET show low metabolism
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 Sprague–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 diagnosed with refractory epilepsy by neurologists according to the definition of pharmacoresistant epilepsy [12]. The 24-h EEG monitoring indicated that epilepsy-like waves originated 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) cortex, so that normal temporal cortex tissues were used as
123
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 properties [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
Neurol Sci 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 Reverse TaqMan probe
WB 50 -GCCGAAAACATTCGCTATG-30 50 -TCTCACCAACCAGGGTGT-30 50 -CTGTCAAGGAAGCCAATGCC-30
b-Actin Primer Forward
50 -AAGGCCAACCGCGAGAA-30
Reverse
50 -CCTCGTAGATGGGCACA-30
TaqMan probe
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).
50 -CTGCACCACCAACTGCTTAGC-30
for 1 week before the start of the experiment. CL is an epileptogenic 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. Seizures 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.
Tissues samples (100 mg) were homogenized and centrifuged. 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 (Amersham 80-6418-77, USA) and wet transferred to PVDF membrane (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). The b-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 compared 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 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 performed 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.
In the human brain tissue samples, the R value of HIF-1a mRNA was 2.91 in the hippocampus and 3.39 in the MTL cortex versus the control cortex, with significant differences, 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.
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Neurol Sci 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
In the rat samples, the expression rates of HIF-1a mRNA 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.
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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
Neurol Sci 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
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) compared 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 epileptogenic focus [17]. On the other hand, numerous studies, in the tumor pharmacoresistance mechanism, have demonstrated 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-regulated in hippocampus and MTL cortex/entorhinal cortex. The expression of MDR1 in both rat and human are positively 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 electrographic seizure discharges originated from MTL can lead to enhanced local oxygen consumption and oxygen desaturations in whole body [20, 21], resulting in HIF-1a accumulation 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 manifests dual nature of apoptosis and adaptive [28]. Considering 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. Moreover, 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.
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Neurol Sci
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-1a may have a regulatory effect on MDR1 expression in pharmacoresistant epilepsy, which is similar to the multidrug 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 Natural 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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12. Kwan P, Arzimanoglou A, Berg AT et al (2010) Definition of drug resistant epilepsy: consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies. Epilepsia 51(6):1069–1077 13. Wang Y, Zhou D, Wang B et al (2003) A kindling model of pharmacoresistant temporal lobe epilepsy in Sprague–Dawley rats induced by coriaria lactone and its possible mechanism. Epilepsia 44(4):475–488 14. Zhou H, Tang YH, Zheng Y (2006) A new rat model of acute seizures induced by tutin. Brain Res 1092(1):207–213 15. Racine RJ (1972) Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32(3):281–294 16. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408 17. Feldmann M, Asselin M-C, Liu J, Wang S et al (2013) P-glycoprotein expression and function in patients with temporal lobe epilepsy: a case-control study. Lancet Neurol 12(8):777–785 18. Song X, Liu X, Chi W et al (2006) Hypoxia-induced resistance to cisplatin and doxorubicin in non-small cell lung cancer is inhibited by silencing of HIF-1alpha gene. Cancer Chemother Pharmacol 58(6):776–784 19. Huang LE, Gu J, Schau M et al (1998) Regulation of hypoxiainducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin–proteasome pathway. Proc Natl Acad Sci USA 95(14):7987–7992 20. Maglajlija V, Walker MC, Kovac S (2012) Severe ictal hypoxemia following focal, subclinical temporal electrographic scalp seizure activity. Epilepsy Behav 24(1):143–145 21. Blum AS, Ives JR, Goldberger AL et al (2000) Oxygen desaturations triggered by partial seizures: implications for cardiopulmonary instability in epilepsy. Epilepsia 41(5):536–541 22. Tae WS, Joo EY, Kim JH et al (2005) Cerebral perfusion changes in mesial temporal lobe epilepsy: SPM analysis of ictal and interictal SPECT. Neuroimage 24(1):101–110 23. Oommen KJ, Saba S, Oommen JA et al (2004) The relative localizing value of interictal and immediate postictal SPECT in seizures of temporal lobe origin. J Nucl Med 45(12):2021–2025 24. Vielhaber S, Von Oertzen JH, Kudin AF et al (2003) Correlation of hippocampal glucose oxidation capacity and interictal FDGPET in temporal lobe epilepsy. Epilepsia 44(2):193–199 25. Matheja P, Kuwert T, Ludemann P et al (2001) Temporal hypometabolism at the onset of cryptogenic temporal lobe epilepsy. Eur J Nucl Med 28(5):625–632 26. Eid T, Brines ML, Cerami A et al (2004) Increased expression of erythropoietin receptor on blood vessels in the human epileptogenic hippocampus with sclerosis. J Neuropathol Exp Neurol 63(1):73–83 27. Rigau V, Morin M, Rousset MC et al (2007) Angiogenesis is associated with blood–brain barrier permeability in temporal lobe epilepsy. Brain 130(Pt 7):1942–1956 28. Chen W, Ostrowski RP, Obenaus A et al (2009) Prodeath or prosurvival: two facets of hypoxia inducible factor-1 in perinatal brain injury. Exp Neurol 216(1):7–15
ORIGINAL ARTICLE
Expression of HIF-1a and MDR1/P-glycoprotein in refractory mesial temporal lobe epilepsy patients and pharmacoresistant temporal 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 Ó 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 epilepsy (mTLE) patients that underwent surgery, and established 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/entorhinal cortex of mTLE patients and the pharmacoresistant TLE rat model. HIF-1a may have a regulatory effect on MDR1 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: [email protected] D. Zhou e-mail: [email protected] J. Chen D. Lei Department of Neurosurgery, West China Hospital, Sichuan University, No. 37 Wainan Guoxue Road, Chengdu 610041, Sichuan, China
expression in refractory epilepsy, which is probably consistent 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-glycoprotein (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 functional HIF-1a binding site known as classical hypoxia response element (HRE) [10]. Recurrent seizures and frequent epileptic discharges may also cause ambient hypoxia, resulting in HIF-1a accumulation to adapt to hypoxic environments. Considering those evidences, we hypothesized that HIF1a 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,
123
Neurol Sci 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 2
M
49
Operative route of benign neoplasm in deep area of brain
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 PET a
Indicates PET show low metabolism
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 Sprague–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 diagnosed with refractory epilepsy by neurologists according to the definition of pharmacoresistant epilepsy [12]. The 24-h EEG monitoring indicated that epilepsy-like waves originated 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) cortex, so that normal temporal cortex tissues were used as
123
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 properties [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
Neurol Sci 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 Reverse TaqMan probe
WB 50 -GCCGAAAACATTCGCTATG-30 50 -TCTCACCAACCAGGGTGT-30 50 -CTGTCAAGGAAGCCAATGCC-30
b-Actin Primer Forward
50 -AAGGCCAACCGCGAGAA-30
Reverse
50 -CCTCGTAGATGGGCACA-30
TaqMan probe
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).
50 -CTGCACCACCAACTGCTTAGC-30
for 1 week before the start of the experiment. CL is an epileptogenic 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. Seizures 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.
Tissues samples (100 mg) were homogenized and centrifuged. 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 (Amersham 80-6418-77, USA) and wet transferred to PVDF membrane (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). The b-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 compared 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 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 performed 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.
In the human brain tissue samples, the R value of HIF-1a mRNA was 2.91 in the hippocampus and 3.39 in the MTL cortex versus the control cortex, with significant differences, 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.
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Neurol Sci 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
In the rat samples, the expression rates of HIF-1a mRNA 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.
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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
Neurol Sci 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
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) compared 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 epileptogenic focus [17]. On the other hand, numerous studies, in the tumor pharmacoresistance mechanism, have demonstrated 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-regulated in hippocampus and MTL cortex/entorhinal cortex. The expression of MDR1 in both rat and human are positively 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 electrographic seizure discharges originated from MTL can lead to enhanced local oxygen consumption and oxygen desaturations in whole body [20, 21], resulting in HIF-1a accumulation 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 manifests dual nature of apoptosis and adaptive [28]. Considering 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. Moreover, 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.
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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-1a may have a regulatory effect on MDR1 expression in pharmacoresistant epilepsy, which is similar to the multidrug 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 Natural 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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