Drion et al. Journal of Neuroinflammation (2018) 15:212
https://doi.org/10.1186/s12974-018-1247-9

RESEARCH

Open Access

Effects of rapamycin and curcumin on
inflammation and oxidative stress in vitro
and in vivo — in search of potential antiepileptogenic strategies for temporal lobe
epilepsy
C. M. Drion1, J. van Scheppingen2, A. Arena3, K. W. Geijtenbeek2, L. Kooijman1, E. A. van Vliet1,2, E. Aronica2,4
and J. A. Gorter1*

Abstract
Background: Previous studies in various rodent epilepsy models have suggested that mammalian target of rapamycin(mTOR) inhibition with rapamycin has anti-epileptogenic potential. Since treatment with rapamycin produces unwantedside effects, there is growing interest to study alternatives to rapamycin as anti-epileptogenic drugs. Therefore, weinvestigated curcumin, the main component of the natural spice turmeric. Curcumin is known to have anti-inflammatory
and anti-oxidant effects and has been reported to inhibit the mTOR pathway. These properties make it a potential antiepileptogenic compound and an alternative for rapamycin.
Methods: To study the anti-epileptogenic potential of curcumin compared to rapamycin, we first studied the effects ofboth compounds on mTOR activation, inflammation, and oxidative stress in vitro, using cell cultures of human fetal
astrocytes and the neuronal cell line SH-SY5Y. Next, we investigated the effects of rapamycin and intracerebrally applied
curcumin on status epilepticus (SE)—induced inflammation and oxidative stress in hippocampal tissue, during early stagesof epileptogenesis in the post-electrical SE rat model for temporal lobe epilepsy (TLE).
Results: Rapamycin, but not curcumin, suppressed mTOR activation in cultured astrocytes. Instead, curcumin suppressed
the mitogen-activated protein kinase (MAPK) pathway. Quantitative real-time PCR analysis revealed that curcumin, but not
rapamycin, reduced the levels of inflammatory markers IL-6 and COX-2 in cultured astrocytes that were challenged with IL1β. In SH-SY5Y cells, curcumin reduced reactive oxygen species (ROS) levels, suggesting anti-oxidant effects. In the post-SE
rat model, however, treatment with rapamycin or curcumin did not suppress the expression of inflammatory and oxidative
stress markers 1 week after SE.
Conclusions: These results indicate anti-inflammatory and anti-oxidant properties of curcumin, but not rapamycin, in vitro.
Intracerebrally applied curcumin modified the MAPK pathway in vivo at 1 week after SE but failed to produce antiinflammatory or anti-oxidant effects. Future studies should be directed to increasing the bioavailability of curcumin(or related compounds) in the brain to assess its anti-epileptogenic potential in vivo.
Keywords: Rapamycin, Curcumin, Inflammation, Oxidative stress, Temporal lobe epilepsy, mTOR, MAPK* Correspondence: j.a.gorter@uva.nl
1
Center for Neuroscience, Swammerdam Institute for Life Sciences, Universityof Amsterdam, Amsterdam, The Netherlands
Full list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Drion et al. Journal of Neuroinflammation (2018) 15:212

BackgroundTemporal lobe epilepsy (TLE) is the most common formof acquired epilepsy in adults [1]. TLE is characterized
by progressive development of spontaneous seizures
after an initial insult, often associated with hippocampal
sclerosis, mossy fiber sprouting, and blood-brain barrier
dysfunction [2]. About 30% of TLE patients do notrespond to treatment with anti-epileptic drugs (AEDs)
that are used to suppress seizures [3, 4]. Therefore, there
is a need to develop treatments that interfere with epileptogenic mechanisms (anti-epileptogenic strategies).
In recent years, the mammalian target of rapamycin(mTOR) pathway has been studied as a possible target
for anti-epileptogenic strategies [5–7]. The mTOR pathway regulates a large number of cellular processes [8, 9],
and mTOR hyperactivation occurs in TLE patients andin several experimental models for epilepsy [10]. Several
studies have shown potential anti-epileptogenic properties of mTOR pathway inhibitor rapamycin in different
experimental models of TLE [11–14].
Still, the mechanisms mediating possible anti-epileptogenic
effects of rapamycin remain to be elucidated. In the electricalpost-status epilepticus (SE) rat model for TLE, where the SE
is the equivalent of the initial insult, it was shown thatchronic treatment with the mTOR inhibitor rapamycin suppressed spontaneous seizures and reduced cell death, mossy
fiber sprouting, and blood-brain barrier leakage after SE [14].
Since rapamycin was able to reduce SE-induced microgliosis
in rats after pilocarpine-induced SE [15] and kainicacid-induced SE [16] and has been shown to be neuroprotective after traumatic brain injury [17, 18] and stroke [19], it
was proposed that rapamycin might have anti-epileptogenic
effects through anti-inflammatory actions, possibly mediatedby inhibition of the mTOR pathway.
However, rapamycin is effective in suppressing epileptogenesis only when treatment is continued, and rapamycin
blood levels remain sufficiently high [20]. Moreover, prolonged treatment with rapamycin produces unwanted side
effects, primarily on growth [14, 20, 21]. Therefore, there
is a growing interest for alternative anti-epileptogenic
treatments through mTOR inhibition. In this context,
curcumin is considered. Curcumin is the main componentof turmeric from the Curcuma longa plant. It is known foranti-inflammatory and neuroprotective properties [22–24], but it has also been reported to inhibit the mTOR
pathway [25] and the mitogen-activated kinase (MAPK)
pathways (extracellular signal-regulated kinase (ERK)1/2
and p38 pathway) [26]. In addition, curcumin has
anti-oxidant effects [23, 27, 28]. No adverse effects of
curcumin have been reported in phase 1 clinical studies[29, 30]. Because of its rapid degradation, curcumin has a
low bioavailability in vivo [31] which could pose a challenge for its use as an anti-epileptogenic drug. Still, its
anti-inflammatory, anti-oxidant, and mTOR-inhibiting

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properties make curcumin potentially anti-epileptogenic
and possibly an interesting alternative to rapamycin.
Here, we aim to elucidate anti-inflammatory andanti-oxidant effects of curcumin compared to rapamycinin the context of epileptogenesis. We first studied the effects of both compounds on inflammation in vitro. Next,
we studied anti-inflammatory and anti-oxidant effects of
rapamycin and curcumin in vivo, in the early phase of
epileptogenesis after SE in rats. With this combined
approach, we aim to shed light on the anti-epileptogenic
potential of curcumin compared to rapamycin and study
the possible anti-inflammatory and anti-oxidant actions
as potential underlying mechanisms.

Methods
Effects of rapamycin and curcumin on inflammation andoxidative stress in vitro

To assess the effects of rapamycin and curcumin on inflammation in vitro, we used primary human fetal astrocyte
cell cultures and studied the levels of pro-inflammatory cytokines after challenging the cultures with interleukin 1-β(IL-1β). To study the effects of curcumin on oxidative
stress in vitro, we studied the reactive oxygen species(ROS) levels in human primary neuronal cultures.
Astrocyte cell cultures

Primary astrocyte-enriched cell cultures were made fromhuman fetal brain tissue (cortex, 14–19 gestational
weeks) obtained from medically induced abortions. A
written informed consent for the use of the tissue forresearch purposes was given by all donors to the Bloemenhove Clinic. The tissue was obtained in accordancewith the Declaration of Helsinki and the Academic Medical Center (AMC) Research Code provided by the Medical Ethics Committee of the AMC. Cell isolation was
performed as described in Additional file 1 and elsewhere [32]. Cultures were incubated with Dulbecco’s
modified Eagle’s medium (DMEM)/HAM F10 (1:1)
medium (Gibco, Life Technologies, Grand Island, NY,
USA), supplemented with 1% penicillin/streptomycin
and 10% fetal calf serum (FCS; Gibco, Life Technologies,
Grand Island, NY, USA). Cultures were refreshed twice aweek and reached confluence after 2–3 weeks. Secondary
astrocyte cultures for experimental manipulation were
established by trypsinizing confluent cultures and re-plating
onto poly-L-lysine (PLL; 15 μg/ml, Sigma-Aldrich; St. Louis,
MO, USA)-precoated 12 and 24-well plates (Costar,Cambridge, MA, USA; 10 × 104 cells/well in a 12-well platefor RNA isolation and quantitative real-time PCR; 5 × 104
cells/well in a 24-well plate for immunocytochemistry). Astrocytes were used for analyses at passages 2–4. Cell cultures were stimulated with human recombinant (r)IL-1β(Peprotech, Rocky Hill, NJ, USA; 10 ng/ml) for 24 h. Treatment of astrocytes with rapamycin (100 nM) or curcuminDrion et al. Journal of Neuroinflammation (2018) 15:212(10 μM) in 0.05% dimethyl sulfoxide (DMSO) was either
started 24 h before, and continued during IL-1β stimulation(pre-treatment), or given simultaneously with IL-1β stimulation (simultaneous treatment). Concentrations of rapamycin (100 nm) and curcumin (10 μM) were selected basedon previous work [33–36], and we selected the concentrations used in the experiments (100 nm rapamycin and10 μM curcumin) after testing multiple concentrations withcell viability assays—see Additional file 1 and Additional
file 2: Figure S1). Cells were harvested 24 h after stimulation with IL-1β.
Human neuronal culture

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Animals

Adult male Sprague Dawley rats (Harlan Netherlands,
Horst, The Netherlands) weighing 250–350 g at the startof the experiment were used. Rats were housed individually in a controlled environment (21 ± 1 °C, 60% humidity, 12-h light/dark cycle with lights on 08:00 AM–
8:00 PM), with water and food (standard laboratorychew) available ad libitum. All animal procedures wereapproved by the Animal Ethics Committee of the
University of Amsterdam, according to Dutch law, andperformed in accordance with the guidelines of theEuropean Community Council Directives 2010/63/EU.

To study the effects of curcumin on oxidative stress,
we used the human neuroblastoma SH-SY5Y cell line,
which is widely used for studying oxidative stress invitro [37]. SH-SY5Y cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM)/HAM F12 (1:1)(Gibco, Life Technologies, Grand Island, NY, USA)supplemented with 1% penicillin/streptomycin and
10% FCS (Gibco, Life Technologies, Grand Island,
NY, USA). The cells were seeded into 96-well cell
culture plates at a density of 10 × 103 cells per welland allowed to adhere for 24 h in a 5% CO2 incubator at 37 °C. The culture medium was then replacedwith either fresh medium containing 0.05% DMSO
alone (vehicle) or with different concentrations of
curcumin (1, 5, 10, and 20 μM) in 0.05% DMSO, andcell cultures were incubated in a 5% CO2 incubatorfor 30 min. For cell viability analysis, see Additional file 1
and Additional file 3: Figure S2).

Status epilepticus induction

Oxidative stress assay

Rapamycin and curcumin treatment

Intracellular ROS levels were measured in SH-SY5Y cells
after treatment with the different concentrations (1, 5,
10, and 20 μM) of curcumin using the 2′7′-dichlorofluorescein (DCF, Sigma-Aldrich, St Louis, MO, USA)method, which is described in Additional file 1. The formation of DCF due to the ROS-driven oxidation of
H2DCFH was measured using a microplate reader withexcitation and emission wavelengths of 485 nm (bandwidth 5 nm) and 535 nm (bandwidth 5 nm), respectively.
In this assay, the levels of DCF fluorescence are directly
proportional to intracellular ROS levels and reported asa fold change compared to the control samples. All
assays were performed in triplicate for each condition.
Effects of rapamycin and curcumin on inflammation in
the post-SE rat model for TLE

To study whether rapamycin and curcumin could beanti-inflammatory in early stages of epileptogenesis, wetested the effects of 1-week treatment with rapamycin or
curcumin following electrically induced SE in rats.

Rats were implanted with intracranial electrodes forstimulation and EEG recording using surgical procedures that are described in Additional file 1 and elsewhere [38]. After several weeks of recovery, the rats
underwent tetanic stimulation of the angular bundle in
the form of a succession of trains of 50-Hz pulses every
13 s. Each train lasted 10 s and consisted of biphasic
pulses of 0.5 ms with a minimal intensity of 300 μA anda maximal intensity of 700 μA. Stimulation was stopped
when the rats displayed sustained forelimb clonus andsalivation for several minutes, which usually occurred
within 1 h. If not, stimulation was continued but neverlasted longer than 110 min. SE was defined electrographically by the occurrence of periodic epileptiformdischarges (PEDs) of 1–2 Hz in the hippocampal EEG
immediately after termination of stimulation. Behavior
was observed during electrical stimulation and several
hours thereafter.

Stock solutions of 150 mg/ml rapamycin (LC Laboratories,
Woburn, MA, USA) were prepared in 100% ethanol andstored at − 20 °C until use. Prior to use, rapamycin stock
was diluted in a vehicle solution (5% Tween 80 + 5% polyethylene glycol 400) resulting in a 4% ethanol containing
solution. Rats (n = 5) were injected intraperitoneally with
6 mg/kg rapamycin solution, starting 4 h after SE and once
daily for 7 days thereafter. A vehicle post-SE group (n = 5)
was injected with vehicle solution following the same paradigm. To allow for intracerebral ventricle (icv) injections
for curcumin treatment, a stainless steel cannula wasplaced on the cortex of the rats during electrode implantation at 1.0 mm AP and 2.5 mm ML from Bregma and secured to the skull with dental cement. Curcumin (70%,
Sigma-Aldrich, Zwijndrecht, The Netherlands) was storedat − 20 °C and freshly prepared (diluted in DMSO) on eachtreatment day. Rats (n = 7) were injected via the icv cannula with 2 μl of 2 mM curcumin solution, and a vehicle
post-SE group (n = 4) was injected with a vehicle solution(DMSO, Sigma) using the same paradigm. Similar to theDrion et al. Journal of Neuroinflammation (2018) 15:212

rapamycin treatment, curcumin injections started 4 h after
SE and continued with daily injections for 7 days after SE.
Based on the estimates of the cerebrospinal fluid (CSF) volume in the rat [39, 40], the end concentration of curcumin
was estimated at a range of ~ 20–40 μM, which is comparable with in vitro levels reported previously [33] and in our
vitro data in the supplementary results (Additional file 3:
Figure S2). The end concentration of DMSO in the cerebrospinal fluid was ~ 1–2%. Control rats were included
that were not stimulated or treated.
One week after SE, the rats were killed by decapitation
under isoflurane anesthesia, and the hippocampal sections were dissected and stored in − 80 °C until use. This
time point was chosen because it coincides with the activation of microglia and astrocytes and the upregulationof various pro-inflammatory markers, according to previous findings [41, 42].
RNA isolation and quantitative real-time quantitative PCR

For RNA isolation, cell culture material was homogenizedin Qiazol Lysis Reagent (Qiagen Benelux, Venlo, The
Netherlands). Total RNA was isolated using the miRNeasy
Mini kit (Qiagen Benelux, Venlo, The Netherlands)
according to the manufacturer’s instructions. For rat hippocampal tissue sections, total RNA was isolated using
the TRIzol® LS Reagent, following the manufacturer’s instructions (Invitrogen—Life Technologies, Breda, The
Netherlands). Concentration and purity of RNA were determined at 260/280 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE,
USA). To evaluate mRNA expression, 200–500 ng (for cell
culture-derived total RNA) or 2500 ng (for rat hippocampal total RNA) was reverse-transcribed into cDNA using
oligo dT primers. Quantitative RT-PCRs were run on a
Roche Lightcycler 480 thermocycler (Roche Applied
Science, Basel, Switzerland) using the following primers:for human cell cultures: IL-6 (forward: ctcagccctgagaaaggaga; reverse: tttcagccatctttggaagg), COX-2 (forward:
gaatggggtgatgagcagtt; reverse: gccactcaagtgttgcacat), EF1a(forward: atccacctttgggtcgcttt; reverse: ccgcaactgtctgtctcatatcac), and C1orf43 (forward: gatttccctgggtttccagt; reverse: attcgactctccagggttca). For rat hippocampal samples:
IL-1β (forward: aaaaatgcctcgtgctgtct; reverse: tcgttgctt
gtctctccttg), IL-6 (forward: gccagagtcattcagagcaa; reverse:
cattggaagttggggtagga), TGF-β (forward: cctggaaagggctcaac
ac; reverse: cagttcttctctgtggagctga), Hmox-1 (forward: caa
ccccaccaagttcaaaca; reverse: aggcggtcttagcctcctctg), HM
GB-1 (forward: gtaattttccgcgcttttgt; reverse: tcatccagga
ctcatgttcagt), cyclophilin A (CycA) (forward: cccaccgtgt
tcttcgacat; reverse: aaacagctcgaagcagacgc), and GAPDH(forward: atgactctacccacggcaag; reverse: tactcagcaccagcatc
acc).
Quantification of data was performed using the computer program LinRegPCR in which linear regression on

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the Log (fluorescence) per cycle number data is applied
to determine the amplification efficiency per sample[43]. The starting concentration of each specific productwas divided by the geometric mean of the starting concentration of the reference genes (EF1a and C1orf43 forcell cultures and CycA or GAPDH for hippocampal
sections), and this ratio was compared between groups.
For the in vivo experiments, ratios were normalized to
not-stimulated control group values. If treatment groups
were not different from their corresponding vehicle
group, the vehicle and treatment groups were pooled tomake one post-SE group to compare to the corresponding not-stimulated control group.
Western blot

Cells in culture or rat hippocampal tissue sections were
homogenized in lysis buffer containing 10 mM Tris (pH8.0), 150 mM NaCl, 10% glycerol, 1% NP-40, 0.4 mg/ml
Na-orthovanadate, 5 mM EDTA (pH 8.0), 5 mM NaF,
and protease inhibitors (cocktail tablets, Roche Diagnostics, Mannheim, Germany). Protein content was determined using the bicinchoninic acid method. For
electrophoresis, an equal amount of proteins (10 or
15 μg/lane for cell cultures and rat hippocampal sections, respectively) were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE,
12% acrylamide). Separated proteins were transferred tonitrocellulose paper by electroblotting for 1 h and
30 min (BioRad, Transblot SD, Hercules, CA, USA).
After blocking for 1 h in TBST (20 mM Tris, 150 mM
NaCl, 0.1% Tween, pH 7.5)/5% non-fat dry milk, blots
were incubated overnight at 4 °C with the primary antibodies (phosphorylated S6 (pS6) #5364, rabbit monoclonal, Cell Signaling Technology, Danvers, MA, USA,
1:1000; MAPK #9102, rabbit monoclonal, Cell Signaling
Technology, 1:1500; phosphorylated MAPK (pMAPK)#4370, rabbit monoclonal, Cell Signaling Technology,
1:1500; β-tubulin, mouse monoclonal, Sigma, St. Louis,
MO, USA; 1: 50,000). After several washes in TBST, themembranes were incubated in TBST/5% non-fat dry
milk, containing the goat anti-rabbit or rabbit
anti-mouse antibodies coupled to horseradish peroxidase(1:2500; Dako, Glostrup, Denmark) for 1 h. After washes
in TBST, immunoreactivity was visualized using ECLWestern blotting detection reagent (Thermo Fisher Scientific, Wilmington, DE, USA). For the culture experiments, for each condition, two wells were analyzed froma total of two donors.
Data analysis

Data were analyzed using non-parametric testing (Kruskal-Wallis followed by Dunn’s tests for multiple comparisons,or Mann-Whitney U tests), using GraphPad™ Prism v.5

Drion et al. Journal of Neuroinflammation (2018) 15:212(GraphPad Software, Inc. La Jolla, CA, USA). p < 0.05 wasassumed to indicate a significant difference.

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a

b

c

d

Results
Effects of curcumin and rapamycin on inflammation andoxidative stress in vitro

Anti-inflammatory effects of rapamycin and curcumin
were tested in vitro by measuring COX-2 and IL-6
mRNA expression in astrocyte cultures that were challenged with IL-1β stimulation (Fig. 1). IL-1β stimulation
increased the expression of COX-2 and IL-6 in cultured
astrocytes (p < 0.005 for both markers). The increase in
IL-6 could be reduced by 10 μM curcumin, both withpre-treatment and simultaneous treatment (p < 0.01),
whereas rapamycin did not affect the IL-1β-induced
increase in IL-6 expression. The IL-1β-induced increase
in COX-2 expression was reduced by simultaneous curcumin (10 μM) treatment (p < 0.005), but not by
pre-treatment. Rapamycin (both pre- and simultaneous
treatment with 100 nM) further increased the expressionof COX-2, compared to IL-1β stimulation (p < 0.005).
To investigate the involvement of the mTOR pathway,
the effect of rapamycin (100 nM) and curcumin (10 μM)
on the downstream target of mTOR, phosphorylated S6(pS6), was evaluated by Western blot (Fig. 2). Rapamycin, but not curcumin, suppressed pS6 levels in cultured
astrocytes, in the presence of IL-1β, and without IL-1β
stimulation. We next investigated whether curcumin
could suppress the MAPK (ERK1/2) pathway, using
Western blot for phospho-(p)MAPK (ERK1/2) andfound that curcumin (10 μM) suppressed the levels of
pMAPK in cultured astrocytes (Fig. 3).
Next, we assessed the anti-oxidant effects of curcuminin vitro, by studying reactive oxygen species (ROS) in
human SH-SY5Y cell cultures using a DCF assay. Curcumin (10 and 20 μM) reduced ROS levels after 30 min
acute treatment compared to control (10 μM, p < 0.05;
20 μM, p < 0.001, Fig. 4).
Effects of curcumin and rapamycin on inflammation andoxidative stress in post-SE rats

We then set out to test the effects of rapamycin and curcumin in vivo, in the post-SE rat model for TLE. Rapamycin (6 mg/kg) and curcumin (~ 40 μM, see the“Methods” section) treatment was given for 1 week,
starting 4 h after SE. Gene expression of inflammatory
markers IL-1β, IL-6, TGF-β, Hmox-1, and HMGB-1 inthe hippocampus was analyzed using quantitative
RT-PCR 1 week after SE (Fig. 5). The expression of
IL-1β, IL-6, and TGF-β was increased 1 week after SE
compared to non-stimulated control rats. Expression of
IL-1β, IL-6, and TGF-β was not different between treatment (rapamycin or curcumin) and their corresponding
vehicle groups. Expression of HMGB-1 was not different

Fig. 1 Anti-inflammatory effects of curcumin in cultured astrocytesthat were challenged with IL-1β. a Rapamycin treatment did not affectthe IL-1β-induced increase in IL-6 expression. b Curcumin, both before(pre-treatment) and simultaneous treatment with the IL-1β challenge,
reduced the increase in IL-6 expression. c Rapamycin further increased
the expression of COX-2 both after pre-treatment and simultaneous
treatment. d Curcumin simultaneous treatment reduced the IL1β-induced increase of COX-2 expression. Data are normalized to
the IL-1β-stimulated condition (white bars) and shown as mean
± SEM. Pre = pre-treatment, sim = simultaneous treatment, **p <0.01, ***p < 0.001 compared to the IL-1β-stimulated condition

between not-stimulated control rats and post-SE rats.
Expression of Hmox-1 was increased 1 week after SE
compared to non-stimulated control rats. Levels ofHmox-1 expression were not different between treatment (rapamycin or curcumin) groups and their corresponding vehicle groups.
Effect of curcumin on pMAPK levels in post-SE rats

To verify that icv-applied curcumin was able to inhibit
the MAPK pathway in vivo, as was seen in vitro, hippocampal sections of post-SE rats were used forWestern blot analysis. The 42-kDa band of pMAPK(p42) was suppressed in curcumin-treated versus
vehicle-treated rats, whereas the 44-kDa band (p44
MAPK) was enhanced in the curcumin group (Fig. 6)

Drion et al. Journal of Neuroinflammation (2018) 15:212abPage 6 of 11abFig. 2 Effects of rapamycin and curcumin on the activation of mTOR
in cultured astrocytes. a Rapamycin, but not curcumin, reduced theexpression of pS6, indicating that rapamycin suppressed theactivation of the mTOR pathway. b Corresponding Western blot
image, pS6 (32 kDa) and β-tubulin (50 kDa) bands. Pre = pretreatment, sim = simultaneous treatment

indicating that curcumin reached the hippocampus
in sufficient concentrations to influence the MAPK
pathway.

Discussion
We compared anti-inflammatory and anti-oxidant effects of curcumin and rapamycin to investigate whether
curcumin has anti-epileptogenic potential that could
make it an alternative for mTOR inhibitor rapamycin.
We found anti-inflammatory and anti-oxidant effects of
curcumin in vitro but not in vivo in the post-SE rat
model.
When we assessed mTOR inhibition in vitro in theastrocyte cultures, we found that only rapamycin wasable to reduce phosphorylated S6 levels, indicating that
rapamycin, but not curcumin, inhibited the mTOR pathway. Instead, curcumin suppressed pMAPK (ERK1/2) inthe astrocyte cultures. The activity of the MAPK pathway has been associated with different experimentalcFig. 3 Effects of curcumin on activation of MAPK in cultured astrocytes.
a Curcumin decreased the expression of p42 MAPK as well as b p44
MAPK, indicating that curcumin suppressed the activation of the MAPK/ERK pathway. c Corresponding Western blot image, pMAPK (42and 44 kDa) and β-tubulin (50 kDa) bands. Pre = pre-treatment,sim = simultaneous treatment

Drion et al. Journal of Neuroinflammation (2018) 15:212

Fig. 4 Anti-oxidant effects of curcumin in human SH-SY5Y cell cultures.
The relative amount of DCF was dose-dependently reduced by curcumin,
indicating that 10 and 20 μM curcumin reduced ROS in SH-SY5Y cell
cultures. Data are normalized to control (0.05% DMSO—white bar) andshown as mean ± SEM from two separate experiments performed intriplicate (*p < 0.05 compared to control, ***p < 0.001 compared
to control)

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models of epilepsy and seizures [44–50]. Increased
MAPK activity could contribute to cell death [51, 52]
and inflammation [53], and previous studies suggested
that the neuroprotective effects of curcumin could be
mediated by the MAPK pathway [26, 54, 55].
Anti-inflammatory effects differed between rapamycin
and curcumin in the astrocyte cultures. Rapamycin did
not suppress inflammatory cytokine IL-6 expression andeven further increased COX-2 expression after the IL-1βchallenge. This is in line with a previous study in rat
astrocyte cultures that also showed increased COX-2
immunoreactivity [56] in microglia and increased IL-6
production after rapamycin treatment [57]. Since weused IL-1β as a trigger to mimic inflammation, we did
not test rapamycin effects on the Il-1β mRNA level itself,
but an effect by rapamycin on IL-1β and other inflammatory associated proteins (than IL6 and COX-2) cannot
be excluded. Curcumin on the other hand suppressed
IL-6 and COX-2, which is in line with the previously
reported anti-inflammatory effects [23, 54, 55, 58, 59].
Curcumin only suppressed COX-2 when treatment wasapplied simultaneously with the IL-1β challenge. The
findings that curcumin inhibits MAPK pathway and inflammatory cytokines in these experiments support theabFig. 5 Inflammatory or oxidative stress markers were not reduced by rapamycin and curcumin in hippocampal tissue at 1 week after electricallyinduced SE. Expression of inflammatory markers interleukins IL-1β and IL-6, transforming growth factor (TGF)-β, and B oxidative stress marker
Heme oxygenase (Hmox)-1 was increased at 1 week after SE in post-SE rats compared to not-stimulated controls but could not be reduced witha rapamycin or b curcumin treatment. High mobility group box 1 (HMGB-1) expression was not different between control and post-SE groups.
Data are normalized to CycA and shown as mean ± SEM

Drion et al. Journal of Neuroinflammation (2018) 15:212

aPage 8 of 11bcFig. 6 Effects of icv-applied curcumin on activation of the MAPK pathway in rat hippocampus 1 week after SE. a, b Curcumin reduced the expressionof p42 MAPK and increased the expression of p44 MAPK, indicating that curcumin modified the activation of the MAPK/ERK pathway. c Corresponding
Western blot image, pMAPK (42 and 44 kDa) and β-tubulin (50 kDa) bands (*p < 0.05 compared to vehicle)

idea that curcumin could have anti-inflammatory effects
mediated by the MAPK pathway in astrocytes, as suggested by previous studies [54, 55].
In SH-SY5Y cell cultures, we found anti-oxidant effectsof curcumin, as shown by reduced ROS levels.
Anti-oxidant effects of curcumin are likely mediated via
induction of Heme oxygenase (Hmox-1), which can protect against damage by free radicals and programmed
cell death [60]. Curcumin is a known inducer ofHmox-1 [27, 28, 61], which could contribute to its neuroprotective effects.
We then tested whether curcumin and rapamycin
could interfere with inflammation during early epileptogenesis by measuring the expression of several inflammatory markers (after treatment during the first week
after SE). Reduced inflammation would explain the
seizure modifying effects of rapamycin observed later
during epileptogenesis and would be in line with theanti-inflammatory actions of curcumin. At 1 week after
electrically induced SE, there is an increase of activationof microglia and astrocytes, which is accompanied by
the upregulation of various pro-inflammatory markers[41, 42]. Accordingly, we found that mRNA levels of inflammatory cytokines IL-1β, IL-6, and TGF-β wereincreased at this time point in post-SE rats compared tocontrol rats. Upregulation of Hmox-1, indicative of oxidative stress, was also increased at 1 week after SE. We
did not detect any effect of rapamycin or curcumin on
inflammatory and oxidative stress markers at 1 week

after SE. For rapamycin, this is in line with our findingsin vitro. However, the lack of anti-inflammatory effects
in vivo could also be explained by the recent findingsthat rapamycin can contribute to blood-brain barrierleakage at this early time point (but not in the chronic
phase) after SE [16]. Blood-brain barrier leakage could
initially contribute to the enhanced levels of inflammatory markers since it is known that peripheral immune
cells and serum proteins that enter the central nervous
system enhance inflammatory responses [62–64]. Alternatively, starting treatment 4 h after SE could have been
too late for both curcumin and rapamycin to exertanti-inflammatory effects.
In another study using pentylenetetrazole (PTZ)-induced model for chronic epilepsy, curcumin did haveanti-inflammatory and had anti-oxidant effects after oral
administration [65, 66]. In the electrical post-SE rat model,
however, we have not been able to detect effects of curcumin on the exhibition of spontaneous seizures when we
administered curcumin orally [20]. Curcumin is known tohave a low bioavailability due to rapid systemic degradation [31]. To circumvent these problems in the current
study, we administered curcumin intracerebrally andfound that curcumin did influence pMAPK at 1 week after
SE but was not able to suppress brain inflammation. Possibly, SE induces too severe damage to be restored by curcumin treatment, or the dosing, location, and timing of
the curcumin treatment were not sufficient to reverse theSE-induced inflammation and oxidative stress.

Drion et al. Journal of Neuroinflammation (2018) 15:212

Conclusions
Taken together, we found that 1-week post-SE treatment with
rapamycin or curcumin did not alter SE-induced upregulationof markers of inflammation and oxidative stress, while invitro, curcumin displayed anti-inflammatory and anti-oxidant
effects. Since these in vitro results are promising, theanti-epileptogenic potential of curcumin deserves further investigation, possibly employing different TLE models and administration strategies to optimize bioavailability.
Additional files
Additional file 1: Supplemental methods and results. (DOCX 46 kb)
Additional file 2: Figure S1. Effects of IL-1β, rapamycin, and curcuminon the viability of astrocyte cell cultures. (PDF 1690 kb)
Additional file 3: Figure S2. Cell viability of SH-SY5Y cells using MTT
assay. (PDF 219 kb)
Abbreviations
COX-2: Cyclooxygenase 2; DMSO: Dimethylsulfoxide;
EEG: Electroencephalogram; ERK: Extracellular signal-regulated kinase; H2DCFDA: 2′,7′-Dichlorofluorescein diacetate; HMGB-1: High mobility box group 1;
Hmox-1: Heme oxygenase 1; icv: Intracerebroventricular; IL-6/IL1β: Interleukin 6/IL-β; MAPK: Mitogen-activated protein kinase;mTOR: Mammalian target of rapamycin; ROS: Reactive oxygen species;
SE: Status epilepticus; TGF-β: Transforming growth factor β; TLE: Temporal
lobe epilepsy
Acknowledgements
We acknowledge the HIS Mouse Facility of the Academic Medical Center,
Amsterdam, and the Bloemenhove Clinic (Heemstede, The Netherlands) for
providing the fetal tissues. We also acknowledge Grazia Forte and Anand Iyer
for their contributions at the beginning of the experiment.
Funding
The research leading to these results has received funding from the Dutch
Epilepsy Foundation, project number EF 14-08 (CD, JAG) and the European
Union’s Seventh Framework Programme (FP7/2007-2013) under grant
agreement no. 602391 (EPISTOP; JvS, EA) and no. 602102 (EPITARGET; EA,
JAG, EAvV).
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Authors’ contributions
Experiments with astrocyte cultures were performed by JvS and KG.
Experiments with SY-SY5Y cells were performed by AA. The post-SE
experiments were carried out by CD, LK, and EAvV. qPCR of the post-SE
material was performed by CD and LK, and the Western blot was performedby CD. The data was analyzed by KG, JvS, AA, and CD. CD drafted andprepared the manuscript. JG and EA supervised the study and participated in
the experiment design, coordination, and manuscript writing. EAvV, JG, andEA revised and contributed to the manuscript. All authors read and approved
the final manuscript.
Ethics approval
Tissue was obtained in accordance with the Declaration of Helsinki and theAcademic Medical Center (AMC) Research Code provided by the Medical Ethics
Committee of the AMC. The material was collected from donors from whom awritten informed consent for the use of the tissue for research purposes was
obtained by the Bloemenhove Clinic (Heemstede, The Netherlands). All animal
procedures were approved by the Animal Ethics Committee of the University of
Amsterdam, according to Dutch law, and performed in accordance with theguidelines of the European Community Council Directives 2010/63/EU.

Page 9 of 11

Consent for publication
Not applicableCompeting interests
The authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.
Author details
1
Center for Neuroscience, Swammerdam Institute for Life Sciences, Universityof Amsterdam, Amsterdam, The Netherlands. 2Department of (Neuro)
Pathology, Amsterdam UMC, University of Amsterdam, Amsterdam, The
Netherlands. 3Department of Biochemical Sciences, Sapienza University ofRome, Rome, Italy. 4Stichting Epilepsie Instellingen Nederland (SEIN),
Heemstede, The Netherlands.
Received: 12 April 2018 Accepted: 2 July 2018

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