Curcumin Attenuates Colistin-Induced Neurotoxicity in N2a Cells via Anti-inflammatory Activity, Suppression of Oxidative Stress, and Apoptosis

Abstract

Neurotoxicity is an unwanted side-effect seen in patients receiving therapy with the “last-line” polymyxin antibiotics. This is the first study to show that colistin-induced neurotoxicity in neuroblastoma-2a (N2a) cells gives rise to an inflammatory response involving the IL-1β/p-IκB-α/NF-κB pathway. Pretreatment with curcumin at 5, 10, and 20 μM for 2 h prior to colistin (200 μM) exposure for 24 h, produced an anti-inflammatory effect by significantly down-regulating the expression of the pro-inflammatory mediators cyclooxygenase-2 (COX-2), phosphorylation of the inhibitor of nuclear factor-kappa B (NF-κB) (p-IκB)-α, and concomitantly NF-κB levels. Moreover, curcumin significantly decreased intracellular reactive oxygen species (ROS) production and increased the activities of the anti-ROS enzymes superoxide dismutase, catalase, and the intracellular levels of glutathione. Curcumin pretreatment also protected the cells from colistin-induced mitochondrial dysfunction, caspase activation, and subsequent apoptosis. Overall, our findings demonstrate for the first time, a potential role for curcumin for treating polymyxin-induced neurotoxicity through the modulation of NF-κB signaling and its potent anti-oxidative and anti-apoptotic effects.

Full text

Curcumin Attenuates Colistin-Induced Neurotoxicity in N2a Cells
via Anti-inflammatory Activity, Suppression of Oxidative Stress,
and Apoptosis
Chongshan Dai
1
&Giuseppe D. Ciccotosto
2
&Roberto Cappai
2
&Shusheng Tang
1
&
Daowen Li
1
&Sanlei Xie
1
&Xilong Xiao
1
&Tony Velkov
3
Received: 20 May 2016 / Accepted: 30 October 2016
#Springer Science+Business Media New York 2016
Abstract Neurotoxicity is an unwanted side-effect seen in
patients receiving therapy with the Blast-line^polymyxin an-
tibiotics. This is the first study to show that colistin-induced
neurotoxicity in neuroblastoma-2a (N2a) cells gives rise to an
inflammatory response involving the IL-1β/p-IκB-α/NF-κB
pathway. Pretreatment with curcumin at 5, 10, and 20 μMfor
2 h prior to colistin (200 μM) exposure for 24 h, produced an
anti-inflammatory effect by significantly down-regulating
the expression of the pro-inflammatory mediators
cyclooxygenase-2 (COX-2), phosphorylation of the inhibitor
of nuclear factor-kappa B (NF-κB) (p-IκB)-α, and concomi-
tantly NF-κB levels. Moreover, curcumin significantly de-
creased intracellular reactive oxygen species (ROS) produc-
tion and increased the activities of the anti-ROS enzymes su-
peroxide dismutase, catalase, and the intracellular levels of
glutathione. Curcumin pretreatment also protected the cells
from colistin-induced mitochondrial dysfunction, caspase
activation, and subsequent apoptosis. Overall, our findings
demonstrate for the first time, a potential role for curcumin
for treating polymyxin-induced neurotoxicity through the
modulation of NF-κB signaling and its potent anti-oxidative
and anti-apoptotic effects.
Keywords Curcumin .Colistin .Neurotoxicity .
Inflammation .Oxidative stress .Mitochondrial dysfunction
Introduction
Polymyxins (colistin and polymyxin B) are increasingly being
used as last-line therapy to treat infections caused by problem-
atic multidrug-resistant (MDR) Gram-negative pathogens,
such as Acinetobacter baumannii,Pseudomonas aeruginosa,
and Klebsiella pneumoniae [1,2]. An unwanted side-effect of
polymyxin therapy is neurotoxicity [2–4]. Patients receiving
intravenous colistin methanesulfonate (CMS; the inactive pro-
drug of colistin) have been reported to present with neurolog-
ical symptoms such as confusion, dizziness, facial/peripheral
paraesthesia, vertigo, seizures, and less commonly fatal effects
including respiratory muscle weakness, apnoea, and ataxia
[3–6]. Clearly, the development of neuroprotective agents
for co-administration is pivotal to improve the clinical utility
of these very important last-line antibiotics.
Curcumin (diferuloylmethane, a yellow pigment in spice
turmeric) is a natural product polyphenol extracted from the
rhizome of Curcuma longa (Fig. 1a). Apart from its culinary
applications in Indian cooking, it is well known that curcumin
possesses many beneficial pharmacological properties such as
anti-tumor, anti-inflammatory, and anti-oxidant activities
[7–9]. The anti-inflammatory effects of curcumin are mediat-
ed through its ability to suppress pro-inflammatory cytokines
such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-
Electronic supplementary material The online version of this article
(doi:10.1007/s12035-016-0276-6) contains supplementary material,
which is available to authorized users.
*Xilong Xiao
xiaoxl@cau.edu.cn
*Tony Velkov
Tony.Velkov@monash.edu
1
College of Veterinary Medicine, China Agricultural University, 2
Yuanmingyuan West Road, Beijing 100193, People’sRepublicof
China
2
Department of Pathology, Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne,
Parkville, VIC, Australia
3
Drug Delivery, Disposition and Dynamics, Monash Institute of
Pharmaceutical Sciences, Monash University, 381 Royal Parade,
Parkville, VIC 3052, Australia
Mol Neurobiol
DOI 10.1007/s12035-016-0276-6

Mol Neurobiol

6, pro-inflammatory enzymes (e.g., cyclooxygenase 2 (COX-
2)) and inflammatory transcription factors such as NF-κB
[10]. Moreover, curcumin exerts a cyto-protective anti-oxi-
dant action by suppressing the accumulation of damaging
ROS and the subsequent cellular apoptosis [9,11–13].
Given that inflammation plays a key role in many disease
states, curcumin has been investigated as a therapeutic agent
for cancer, cardiovascular, neurological, autoimmune, and
metabolic diseases [10,11]. Notably, curcumin can cross the
blood-brain barrier, indicating a potential application as a neu-
roprotective agent [10,12,14]. Clearly, curcumin’santi-
inflammatory and anti-oxidative properties hold a great deal
of potential for its clinical utility in a large number of chronic
disease states. The primary aim of the present study was to
investigate the neuroprotective pathways of curcumin against
colistin-induced neurotoxicity using a mouse neuroblastoma-
2a (N2a) cell culture model. We also explored the mechanisms
by which curcumin suppresses oxidative stress, mitochondrial
dysfunction, and apoptosis in N2a neuronalcells. The present-
ed findings are discussed in context of the clinical potential of
curcumin as a co-administered neuroprotective agent during
polymxyin therapy.
Materials and Methods
Chemicals
Colistin (sulfate, 20,400 units/mg) was obtained from
Zhejiang Shenghua Biology Co., Ltd. (Zhengjiang, China).
Curcumin and bisdemethoxycurcumin (BDMC; both purity,
≥98 %) and was purchased from Aladdin Reagent Co., Ltd.
(Shanghai, China). 3-(4,5-dimetylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT), dimethyl sulfoxide
(DMSO), and sodium dodecylsulfonate (SDS) were pur-
chased from AMRESCO Inc. (Solon, OH, USA).
Rhodamine (Rh) 123 and 2′,7′-dichlorofluorescensin diacetate
(DCFH-DA) were purchased from Beyotime (Haimen,
China). Dulbecco’s modified Eagle’smedium(DMEM)and
fetal bovine serum (FBS) were obtained from Life
Technologies Corporation (Grand Island, NY, USA). All other
reagents were of highest analytical grade available.
Cell Culture
The mouse N2a cells (ATCC CCL-131™)wereculturedin
DMEM medium supplemented with 10 % (v/v) FBS, 110 mg/
L sodium pyruvate, 100 units/mL penicillin, and 100 μg/mL
streptomycin (Beyotime, Haimen, China) at 37 °C in 5 %
CO
2
. The media were changed once per day.
Mouse primary cortical neurons were prepared from C57/
Bl6 embryonic day 14 mice under sterile conditions using
procedures as previously described and approved by the
Melbourne University Animal Ethics Committee [15].
Briefly, embryonic day 14 C57/BL6 mice cortices were re-
moved, dissected free of meninges, and dissociated in
0.025 % (w/v) trypsin in Krebs buffer. The dissociated cells
were triturated using a filter-plugged fine pipette tip, pelleted,
resuspended in plating medium (minimum Eagle’smedium
containing 10 % fetal calf serum and 5 % horse serum), and
counted. Cortical neuronal cells were seeded at 150,000 cells/
well onto a poly-D-lysine-coated 48-well plate in plating me-
dium for 2 h then replaced with freshly prepared neurobasal
medium containing B27 supplements, gentamicin, and
0.5 mM glutamine (all tissue culture reagents were purchased
from Invitrogen (Australia) unless otherwise stated). The neu-
ronal cells were allowed to mature for 7 days in culture before
commencing treatment using freshly prepared primary culture
media (neurobasal medium plus B27 supplements (minus an-
ti-oxidants). All cultures were maintained in an incubator set
at 37 °C with 5 % CO
2
. This method resulted in cultures
highly enriched for neurons (>95 % purity) with minimal as-
trocyte and microglial contamination. The accumulation of
colistin in primary mouse cortical neurons was visualized
using an anti-polymyxin B mouse IgM antibody (Thermo
Fisher Scientific, Rockford, USA) as previously described
with minor modifications to the protocol [16,17]. Briefly,
the mAb was diluted to 1:500 and incubated with the
colistin-treated cells overnight at 4 °C. The cells were washed
and incubated with M.O. M. biotinylated anti-mouse second-
ary link (Vector Labs, CA, USA) for 10 min, followed by
incubation with an AlexaFluor647 Streptavidin conjugate at
a 1:500 dilution (Life Technologies, VIC, Australia).
Measurement of Cell Viability
Cell viability was measured using the MTT assay. Briefly, N2a
cells (1 × 10
4
) were seeded into 96-well tissue culture plates.
After culture for 12 h, cells were pretreated with different
doses of colistin (50–800 μM). After 24 h, the medium was
discarded and replaced with 100 μL serum-free DMEM
Fig. 1 Protective effects of curcumin and bisdemethoxycurcumin on
colistin-induced cytotoxicity in N2a neuronal cells. aThe chemical struc-
tures of curcumin and bisdemethoxycurcumin. bDose-dependent toxicity
of colistin on N2a cell viability. c,dImpact of curcumin (Cur) and bis-
demethoxycurcumin (BDMC) pretreatment for 2 h (5, 10, and 20 μM) on
colistin-induced (200 μM) cytotoxicity in N2a cells (24 h incubation). e,f
Etoposide (Eto50;50μM) was used as the positive control treatment. g
The neuroprotective effect of curcumin pretreatment (5, 10, and 20 μM
for 2 h) in mouse primary cortical neurons against colistin-induced
(200 μM) cell death. The cell viability data were normalized and calcu-
lated as a percentage of untreated vehicle control values. hConfocal
fluorescence microscopy images of colistin-treated mouse primary corti-
cal neurons stained with anti-polymyxin monoclonal antibody (green
channel). All cell viability data shown represent as the mean ± SD from
five independent experiments. *p<0.05;**p<0.01—compared with the
untreated control;
#
p<0.05;
##
p<0.01—compared with the colistin or
etoposide treatment
Mol Neurobiol

containing 10 μL MTT (5 mg/mL) and the cells were incu-
bated for 4 h at 37 °C. Finally, the medium was discarded and
100 μL DMSO was added. After incubation for 20 min at
room temperature, the absorbance was read at 570 nm in a
micro-plate reader (Molecular Devices, Sunnyvale, CA,
USA). To assess the cyto-protective effects, cells were
pretreated with curcumin or BDMC (5, 10, or 20 μM) for
2 h. Following 2 h, the medium containing curcumin or
BDMC was discarded and the cells were washed with cold
PBS and incubated with colistin (200 μM) for an additional
24 h and then cell viability was assessed. As a positive control,
N2a cells were treated with etoposide (50 μM) for 24 h with or
without curcumin (or BDMC) pretreatment for 2 h at 37 °C.
Mouse primary cortical neurons were pretreated with
curcumin (5, 10, and 20 μM) or vehicle (DMSO) for 2 h then
media was removed and fresh NB media containing colistin
(200 μM) or vehicle (PBS) was added to the wells and incu-
bated for 24 h. At the conclusion of the experiment, MTT
reagent was added to the treated cultures for 3 h at 37 °C then
culture media removed and formazan by product fully dis-
solved using DMSO. A 100-μL aliquot of the MTT/DMSO
was transferred to a 96-well clear-walled plate and the absor-
bance measured at 570 nm in a FLUOstar Omega plate reader
(BMG Labtech, Vic, Australia). The data were normalized and
calculated as a percentage of untreated vehicle control values.
Measurement of Apoptosis
The apoptosis assay was performed using an Annexin V-FITC
apoptosis detection kit according to the manufacturer’sproto-
col (Vazyme Biotech Co., Ltd., Nanjing, China). Epotoside
(50 μM) was used as positive control treatment. For the flow
cytometric analysis, cells were harvested with 0.25 % trypsin
without EDTA, washed twice with cold PBS, and resuspended
in 500 μL binding buffer supplied by the manufacturer. The
cells were then incubated with 5 μL annexin V-FITC (40 μg/
mL) and 5 μL propidium iodide (40 μg/mL) in the dark for
10 min. Analysis was performed using a BD FACSAria™
flow cytometer (Becton Dickinson, San Jose, CA, USA) set
at an excitation wavelength of 488 nm and an emission wave-
length of 605 nm.
Measurement of Intracellular ROS Generation
Intracellular ROS production was measured using the ROS-
specific fluorescent dye 2,7-dichlorofluorescein diacetate
(DCFH-DA) according to the manufacturers protocol
(Beyotime, Haimen, China). N2a cells were plated into 12-
well plates at a density of 2 × 10
5
cells per well and pretreated
with curcumin at final concentrations of 5, 10, or 20 μMfor
2 h at 37 °C and washed with cold PBS. Cells were then
treated with colistin (200 μM) for 24 h. The control cells were
treated with curcumin at 20 μM per se or the vehicle (0.1 %
DMSO in PBS). After treatment, DCFH-DA (10 μM) was
added into the medium for a further 30 min at 37 °C. The cells
were washed twice with PBS, harvested by trypsinization, and
analyzed for ROS production using a BD FACSAria™flow
cytometer set at an excitation and emission wavelength of 485
and 530 nm.
Measurement of Intracellular Superoxide Dismutase,
Catalase Activities, and Intracellular Glutathione Levels
The superoxide dismutase (SOD), catalase (CAT) activities,
and glutathione (GSH) levels were detected using specific
assay kits according to the manufacturer’s instructions
(Nanjing Jiancheng Co., Ltd., Nanjing, China). In brief, N2a
cells were plated into 6-well plates at a density of 5 × 10
5
cells
per well and pretreated with curcumin (5, 10, or 20 μM) at
37 °C for 2 h. After removing the medium-containing
curcumin, the cells were incubated colistin (200 μM) for
24 h. As a positive control cells were treated with etoposide
(50 μM) for 24 h. The negative control cells were treated with
curcumin (20 μM) or the vehicle (0.1 % DMSO in PBS). Cells
were washed with cold PBS and lysed using the cell lysis
buffer provided by the manufacturer. The cell lysates were
centrifuged at 14,000×gfor 10 min at 4 °C. Supernatants were
collected and assayed for SOD, CAT activities, and GSH
levels. Protein concentrations were quantified using the
BCA protein assay kit (Beyotime, Haimen, China).
Measurement of the Change in Mitochondrial Membrane
Potential (Δψm)
The Δψ
m
was detected using the fluorescent indicator JC-1
(Beyotime, Haimen, China). N2a cells were plated into 12-
well plates at a density of 2 × 10
5
cells per well and pretreated
with curcumin (20 μM) at 37 °C for 2 h, followed by treatment
with colistin (200 μM) for 24 h. Epotoside (50 μM) was used
as positive control treatment. After treatment, N2a cells were
incubated in DMEM containing 10 μM JC-1 at 37 °C for
15 min, washed with PBS, and observed under a fluorescence
microscope (Leica Microsystems, Wetzlar, Germany). A shift
of fluorescence from red to green represents a loss of ψ
m
.JC-1
red fluorescent emission (normal ψ
m
) was measured at
583 nm with an excitation wavelength of 525 nm, and JC-1
green fluorescence emission (loss of ψ
m
) was measured with
an excitation wavelength of 525 and emission wavelength of
530 nm. For quantitative analysis, at least 100 regions of in-
terest were selected in each group and the ratios between fluo-
rescence intensity in the green (low membrane potential) and
red (high membrane potential) channels in each region of
interest were calculated. An increase in the ratio was
interpreted as the loss of ψ
m
.
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Measurement of Caspase-3/7 and Caspase-9 Activities
N2a cells (1.5 × 10
4
cells per well) were cultured in 96-well plates
and treated with curcumin (5, 10, or 20 μM) for 2 h at 37 °C.
After removing the medium-containing curcumin, the cells were
then incubated in media-containing colistin (200 μM)for24hat
37 °C. Epotoside (50 μM) was used as positive control treatment.
The caspase-3/7 and caspase-9 activities were determined using
the Caspase-Glo®-3/7 and caspase-9 assay kits according to the
manufacturer’s instructions (Promega Corp., Madison, USA).
The luminescence was measured using a micro-plate
luminometer (Molecular Devices, Sunnyvale, CA, USA).
Wester n Blo tting
Following treatment, N2a cells were harvested using a scraper
and lysed in 100 mM Tris-HCl, pH 7.4, 2 % (w/v)SDS,10%
(v/v) glycerol, and 1 mM PMSF. Cell lysates were centrifuged
at 14,000×gfor 15 min at 4 °C, and supernatants was subject-
ed to Western blotting analysis. The proteins were resolved
using 10–12 % SDS-PAGE gels and then transferred to a
nitrocellulose membrane (Applygen Technologies Inc.
Beijing, China). The membranes were blocked with 5 % (w/
v) skim-milk powder dissolved in Tris-buffered saline and
Tween 20 (TBST) for 2 h, washed three times with TBST,
and then incubated overnight at 4 °C with rabbit polyclonal
antibodies against COX-2 (ABclonal Biotech Co. Ltd.,
Cambridge, USA), heme oxygenase (HO)-1, Bax, IL-1β,
COX-2, and nuclear factor erythroid 2-related factor 2 (Nrf2;
ProteinTech Group, Inc., USA), p-inhibitor of NF-κB(IκB)-α
(p-IκB-α; Ser32/36; Beijing Biosynthesis Biotechnology Co.,
Ltd., Beijing, China), NF-κB (p65), and glyceraldehyde 3-
phosphate dehydrogenase (GAPDH; all 1: 1000; Santa Cruz
Biotechnology, Inc., USA). After being washed three times
with TBST, the membranes were incubated for 1 h with the
corresponding species-specific horseradish peroxidase-
conjugated secondary antibodies (1:10,000). The blots were
visualized using Western luminescent detection kit (Vigorous
Biotechnology, Beijing, China). The protein load was normal-
ized to GAPDH and quantified using the ImageJ software
(National Institute of Mental Health, Bethesda, MD, USA).
Immunofluorescence Detection of NF-κBProtein
Expression
After treatment, N2a cells were fixed with 4 % paraformalde-
hyde at room temperature for 30 min. The cells were then
washed twice with PBS and then permeabilized with 1 %
Triton X-100 for 20 min. The cells were then incubated in
blocking buffer (2 % bovine serum albumin in PBS) for 2 h
at 37 °C. Cells were incubated with rabbit polyclonal antibod-
ies against NF-κB (1:200; Santa Cruz Biotechnology, Inc.,
USA) overnight at 4 °C, washed twicewith PBS, and followed
by co-incubation with Cy3-labeled goat anti-rabbit IgG sec-
ondary antibodies (Beyotime Institution of Biotechnology,
Haimen, China; both 1:1000) for 2 h at 37 °C. The cells were
then washed twice with PBS and incubated with 4′,6-
diamidino-2-phenylindole nuclear stain for 5 min. The expres-
sion of NF-κB was observed under a fluorescence microscope
at 583 nm with an excitation wavelength of 525 nm.
Quantitative Reverse-Transcription (qRT) PCR
Examination
N2a cells were plated into 6-well culture plates (5 × 10
5
cells
per well) and pretreated with curcumin (5, 10, or 20 μM) at
37 °C for 2 h. After removing the medium-containing
curcumin, the cells were washed with cold PBS and incubated
with colistin (200 μM) for 24 h. The treated cells were har-
vested, and total RNA was isolated using the TRIzol extrac-
tion kit according to the manufacturer’s instructions
(Invitrogen Inc., Carlsbad, CA). The cDNA was synthesized
from ~2 μg of total RNA using the Prime Script RT-PCR kit
(Takara, Dalian, China). The PCR primers used were as fol-
lows: Nrf2: forward, 5′-CAC ATT CCC AAA CAA GAT GC-
3′,reverse,5′-TCT TTT TCC AGC GAG GAG AT-3′;HO-1:
forward, 5′-CGT GCT CGA ATG AAC ACT CT-3′,reverse,
5′-GGA AGC TGA GAG TGA GGA CC-3′; NF-κB: for-
ward, 5′-CAC TGT CTG CCT CTC TCG TCT-3′;reverse,
5′-AAG GAT GTC TCC ACA CCA CTG-3′;GAPDH:for-
ward, 5′-ACA GTC CAT GCC ATC ACT GCC-3′;reverse,
5′-GCC TGC TTC ACC ACC TTC TTG-3′. qRT-PCR were
performed using a Chromo 4™instrument (Bio-Rad,
Hercules, CA), and the cycling conditions used were as fol-
lows: 95 °C for 10 min; 40 cycles of 95 °C for 15 s, 60 °C for
1 min, and 72 °C for 40 s. All reactions were conducted in
triplicate. The fold change in gene expression was calculated
using 2
−ΔΔCt
after normalizing to the expression level of the
reference gene GAPDH.
Statistical Analysis
All data are expressed as the mean ± standard deviation (SD).
Data from the control and treatment groups were analyzed
with one-way analysis of variance, followed by the LSD post
hoc test using SPSS V13.0 (SPSS Inc., Chicago, USA). A p
value <0.05 was considered significant.
Results
Curcumin Attenuates Colistin-Induced Cytotoxicity
of N2a Neuronal Cells
Exposure of N2a cells to colistin produced a dose-dependent
decrease in cell viability (Fig. 1b). For the curcumin cyto-
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protective studies, N2a cells were exposed to 200 μMcolistin
for 24 h, which reduced the cell viability to 47.2 % (p<0.01).
Pretreatment of the N2a cells with curcumin at 5, 10, and
20 μM for 2 h prior to colistin exposure significantly attenu-
ated colistin-induced cytotoxicity and increased the cell via-
bility to 54.7, 63.2, and 73.6 % (all p< 0.01) (Fig. 1c), respec-
tively. Likewise, pretreatment with 5, 10, and 20 μMofthe
curcumin analog BDMC (which accounts for ~17 % of the
composition of curcumin powder) for 2 h, increased N2a cell
viability to 51.4, 56.7 (p< 0.05), and 60.5 % (p<0.01),re-
spectively (Fig. 1d). These findings would suggest that
curcumin has a stronger cyto-protective activity compared
with its congener BDMC. Similarly, in the positive control
etoposide-treated (50 μM) control cells, curcumin showed a
stronger cyto-protective effect compared with BDMC
(Fig. 1e, f). The neuroprotective effect of curcumin (5, 10,
and 20 μM) was validated in cultures of mouse primary
cortical neurons treated with colistin (200 μM) (Fig. 1g).
Curcumin pretreatment at 20 μM effectively protected the
primary cortical neurons from colistin-induced cell death
(>50 % at 200 μM colistin); curcumin treatment per se had
no impact on cell viability. Furthermore, the accumulation of
colistin in the cortical cells was visualized using an anti-
polymyxin monoclonal antibody (Fig. 1h). The imaging
results revealed that colistin accumulates in both the cell
body and the dendrites.
Curcumin Attenuates Colistin-Induced Apoptosis in N2a
Cells
Exposure of N2a cells to 200 μMcolistinfor24hin-
duced apoptotic rates up to 46.4 % (p< 0.01) (Fig. 2a).
Pretreatment of the N2a cells with curcumin at 5, 10, and
20 μM for 2 h prior to colistin exposure decreased the
Fig. 2 Protective effect of curcumin against colistin-induced apoptosis in
N2a cells. aApoptosis of N2a cells were analyzed by flow cytometry
following annexin V-FITV/PI staining. Q1 necrosis cells, Q2 later apo-
ptotic cells, Q3 live cells, Q4 early apoptotic cells. Cells were pretreated
with curcumin for 2 h at 37 °C. After removing the medium-containing
curcumin, the cells were then incubated in media-containing colistin for
24 h at 37 °C. bEtoposide (Eto50;50μM) was used as the positive
control treatment. The data shown represent the mean ± SD, from three
independent experiments. **p<0.01—compared with the untreated con-
trol;
##
p<0.01—compared with the colistin or etoposide treatment
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Mol Neurobiol

apoptotic rates to 42.2, 34.4 (p< 0.01), and 24.5 %
(p< 0.01) (Fig. 2a), respectively. In the positive etoposide
(50 μM) control cells, pretreatment with curcumin
(20 μM) decreased apoptotic rates to 22.6 vs 33.4 % (no
pretreatment) (Fig. 2b). Curcumin treatment per se
(20 μM) had no effect on the apoptotic rates and apoptotic
rates were essentially comparable with untreated control
cells.
Curcumin Attenuates Colistin-Induced Mitochondrial
Dysfunction and Caspase Activation
The neuroprotective effect of curcumin against colistin-
induced mitochondrial dysfunction was assessed using the
JC-1 MTP assay. Colistin treatment (200 μM) for 24 h in-
duced mitochondrial dysfunction in N2a cells seen as an in-
crease of green fluorescent JC-1 (JC-1 monomeric form); the
green/red fluorescence radio increased 3.12 (p<0.01),com-
pared with that in the control (Fig. 3a, b). This indicated the
loss of mitochondrial membrane potential (ψ
m
). Pretreatment
of the N2a cells with 20 μM curcumin for 2 h prior to colistin
exposure protected the N2a cellular ψ
m
against colistin-
induced perturbations, as evidenced by the decreases of the
green/red fluorescence radio (decreased to 0.72; p< 0.01),
compared with the colistin-alone group (Fig. 3a, b). Colistin
exposure at 200 μM for 24 h significantly (all p< 0.01) in-
creased the activities of caspase-9 and caspase-3 to about 2.4-
and 2.8-fold, respectively, compared with the untreated con-
trol cells. Curcumin pretreatment (20 μM), significantly (all
p< 0.01) down-regulated the activation of caspase-9 and
caspase-3 to about 1.5- and 1.7-fold, respectively, compared
with the colistin only treated cells (Fig. 3c, d). In the positive
control etoposide-treated (50 μM) cells, pretreatment with
curcumin (20 μM) significantly (p< 0.05 or 0.01) attenuated
the loss of ψ
m
and down-regulated the activation of caspase-9
and caspase-3 (Fig. 3a–d).
Curcumin Attenuates Colistin-Induced Oxidative Stress
by Decreasing Intracellular ROS, Inducing SOD and CAT
Activities, and Increasing Intracellular GSH Levels
Pretreatment of the N2a cells with curcumin at 5, 10, and
20 μM for 2 h prior to colistin exposure significantly de-
creased the intracellular ROS levels from 227.6 % (colistin
only treatment, to 161.3, 134.6, and 117.8 % (all p<0.01),
respectively (Fig. 4a). Moreover, curcumin pretreatment at
10 and 20 μM significantly increased (p<0.05and
p< 0.01, respectively) the SOD and CAT activities and the
intracellular levels of GSH (Fig. 4b–d). Similarly, in the
positive etoposide (50 μM) control cells, curcumin pretreat-
ment at 20 μM markedly increased the activities of SOD and
CAT and the intracellular levels of GSH (all p<0.05or
0.01) (Fig. 4b–d). Curcumin treatment per se (20 μM) had
no effect on cellular ROS, SOD, and CAT activities and
GSH levels (Fig. 4).
Curcumin down-Regulates the Activation
of Colistin-Induced Pro-inflammatory Mediators,
Pro-apoptotic Proteins, and Nrf2/HO-1 Expression
The protein expression in N2a cells of key pro-inflammatory
mediators including p-IκB-α,NF-κB, IL-1β, COX-2, the pro-
apoptotic protein Bax, and the Nrf2/HO-1 pathway proteins
were examined using Western blotting. Following colistin ex-
posure (200 μM) for 24 h, the expression of p-IκB-α(~18.1-
fold), NF-κB (~16.3-fold), IL-1β(~8.1-fold), COX-2 (~14.7-
fold), Bax (~2.2-fold), Nrf2 (~1.6-fold), and HO-1 (~3.4-
fold), all significantly increased (all p<0.01),comparedwith
the untreated control cells (Fig. 5). Pretreatment of the N2a
cells with curcumin (5, 10, and 20 μM) prior to colistin expo-
sure significantly decreased (p< 0.05 or 0.01) the expression
of p-IκB-α,NF-κB, COX-2, and Bax, in a dose-dependent
manner (Fig. 5). Conversely, curcumin pretreatment increased
the expression of Nrf2 and HO-1 and had no effect on the
expression of IL-1β(Fig. 5). Interestingly, in the positive
etoposide (50 μM) control cells, curcumin pretreatment
(20 μM) decreased the expression of p-IκB-α,NF-κB,
COX-2, and IL-1β(all p< 0.05 or 0.01) and increased the
expression of Nrf2 and HO-1 (Fig. S1). Furthermore, NF-κB
expression in N2a cells in response to colistin exposure de-
tected via immunofluorescence (Fig. 6), showed that
curcumin down-regulates the colistin- or etoposide-induced
expression of NF-κB(Fig.5).
Curcumin Increases Nrf2 and HO-1 mRNA Expression
and Decreases NF-kB mRNA Expression
Exposure of N2a cells to colistin (200 μM) for 24 h signifi-
cantly (all p< 0.05 to <0.01) increased the messenger RNA
(mRNA) expression of Nrf2, OH-1, and NF-κB (Fig. S2).
Fig. 3 Curcumin attenuates colistin-induced mitochondrial dysfunction
and caspase activation. aN2a cells were plated into 12-well plates at a
density of 2 × 10
5
cells per well and pretreated with curcumin at final
concentrations of 20 μM(Cur20)at37°Cfor2h(n= 3). The cells were
treated with or without colistin at 200 μM for additional 24 h. Etoposide
(Eto50;50μM) was used as the positive control treatment. The change in
mitochondrial membrane potential (MTP) was evaluated using the cat-
ionic fluorescent indicator JC-1. The JC-1-aggregate form, indicating
normal MTP function, appears red. The JC-1 monomeric form, indicating
disrupted MTP, appears green. Magnification, ×40. bQuantitative anal-
ysis of the confocal data are presented as the ratio between fluorescence
intensityinthegreen (low membrane potential) and red (high membrane
potential) channels. An increase in the ratio was interpreted as the loss of
ψ
m
.c,dCaspase-9 and caspase-3 activity in N2a cells in response to
colistin and curcumin pretreatment were examined using ELISA. Values
are presented as the mean ± SD, from three independent experiments.
*p<0.05;**p<0.01—compared with the untreated control;
#
p<0.05;
##
p<0.01—compared with the colistin or etoposide treatment
Mol Neurobiol

Pretreatment with curcumin (20 μM) for 2 h markedly de-
creased the expression of NF-κB mRNA and significantly
increased the expression of Nrf2 and OH-1 mRNA, in a
dose-dependent manner (Fig. S2). The expression levels of
the reference gene GAPDH did not change under the condi-
tions used.
Discussion
Neurotoxicity is a major, albeit poorly understood side-effect
associated with polymyxin therapy [3,4,6,18]. We have
previously shown using an in vitro N2a cell culture model that
colistin-induced neurotoxicity involves apoptosis,
Fig. 4 Curcumin protects N2a
cells against colistin-induced oxi-
dative stress. aCellular ROS
levels were detected using flow
cytometry following staining of
N2a cells with the ROS-sensitive
dye 2,7-dichlorofluorescein
diacetate. b–dThe impact of
curcumin pretreatment (5, 10, and
20 μM) for 2 h on cellular super-
oxide dismutase (SOD), catalase
(CAT) activities, and glutathione
(GSH) levels in N2a cells treated
with colistin (200 μM for 24 h).
Etoposide (Eto50;50μM) was
used as the positive control treat-
ment. The data shown represent
the mean ± SD, from three inde-
pendent experiments.
**p<0.01—compared with the
untreated control;
#
p<0.05;
##
p<0.01—compared with the
colistin or etoposide treatment
Mol Neurobiol

mitochondrial dysfunction, generation of ROS, and autopha-
gy [19]. The discovery of neuroprotective agents for co-
administration during colistin therapy is paramount to prolong
the clinical utility of this important last-line lipopeptide anti-
biotic. The aim of the present study was to exploit our in vitro
N2a neuronal cell culture model of colistin-induced neurotox-
icity to study the neuroprotective mechanism of curcumin in
antagonizing pro-inflammatory cytokines by suppressing
NF-κB activation and signaling. We also explored the neuro-
protective molecular mechanisms by which curcumin sup-
press apoptosis, mitochondrial dysfunction, and ROS produc-
tion in the N2a neuronal cells.
Compositionally, curcumin (curry powder) is predominantly
diferuloylmethane (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
heptadiene-3,5 dione) and contains ~17 % bisdemethoxycurcu-
min (BDMC) and 3 % demethoxycurcumin (DMC) [20–22].
Direct curcumin targets include COX-2, lipoxygenase, toll-like
receptor (TLR)4, focal adhesion kinase, glutathione, glycogen
synthase kinase (GSK)-3 β, phosphorylase-3 kinase, xanthine
oxidase, pp60 src tyrosine kinase, and ubiquitin isopeptidase
[23–28]. The molecular targets indirectly regulated by curcumin
include the transcription factors NF-κB, hypoxia inducible factor
(HIF)-1a, activator protein (AP)-1, signal transducers and
activators of transcription protein (STAT)-3, peroxisome
proliferator-activated receptors, p53, anti-apoptotic proteins
(e.g., Bcl-2 and Bcl-xL), cell-cycle regulatory proteins (e.g.,
cyclins D1, E, and c-myc), and inflammatory mediators (e.g.,
IL-1 and IL-6) [10,25,29,30].
In our recent mechanistic toxicity study, we reported that
colistin-induced apoptosis in neuronal N2a cells is activated
via both the death receptor (extrinsic) and the mitochondrial
(intrinsic) pathways [19]. In the present report, we have shown
that colistin-induced apoptosis could be inhibited by pretreat-
ment of the N2a cells with 20 μMofcurcuminfor2h.
Specifically, curcumin pretreatment significantly attenuated the
loss of mitochondrial membrane potential (Fig. 3a) and down-
regulated the expression of the pro-apoptotic protein Bax (Fig. 5)
and the activation of caspase-9 and caspase-3 (Fig. 3b, c).
The nervous system is highly vulnerable to oxidative damage
due to its elevated oxygen demand and high polyunsaturated
fattyacidcontent[31]. We previously reported that colistin-
induced neurotoxicity involves mitochondrial dysfunction in
the mouse cerebral cortex and sciatic nerve tissues in mice in-
travenously injected with 15 mg kg
−1
day
−1
colistin sulfate for
7days[32]. In the present study, we found that colistin exposure
significantly increased intracellular ROS levels in N2a cells with
Fig. 5 Curcumin attenuates colistin-induced expression of pro-
inflammatory cytokines and pro-apoptotic protein and activates the
Nrf2/HO-1 pathway. Left panels, N2a cells treated with colistin
(200 μM for 24 h) with or without curcumin pretreatment (5, 10, and
20 μM for 2 h) were analyzed using western blotting for expression of p-
IκB-α,NF-κB, interleukin (IL)-1β, cyclooxygenase-2 (COX-2), nuclear
factor erythroid 2-related factor 2 (Nrf2; band indicated with asterisk),
heme oxygenase (HO)-1, and Bax. Glyceraldehyde 3-phosphate dehy-
drogenase (GAPDH) was used to demonstrate equal protein loading per
gel lane. Right panels, the relative protein expression levels were ana-
lyzed using ImageJ. Values are presented as mean ± SD, from three
independent experiments. *p<0.05;**p<0.01—compared with the
untreated control;
#
p<0.05;
##
p<0.01—compared with the colistin or
etoposide treatment
Mol Neurobiol

a concomitant decrease in the activity of the anti-oxidant en-
zymes SOD and CAT and decreased intracellular GSH levels
(Fig. 4). Taken together, these findings would suggest that co-
listin neurotoxicity not only induces ROS production directly, it
further decreases the neurons’capacity to breakdown oxygen
radicals, further exacerbating ROS-mediated oxidative stress.
One of the most remarkable pharmacological properties of
curcumin is its anti-oxidant activity, indeed it has been reported
that curcumin can mop up oxygen radicals even more effective-
ly than vitamin E [26]. In the present study, we found that
curcumin could not only inhibit colistin-induced ROS genera-
tion but also enhance the total anti-oxidant capacity in N2a cells
by up-regulating the activities of SOD and CAT and increasing
intracellular GSH levels (Fig. 4). The potent anti-oxidant activ-
ity of curcumin is closely related to its ability to activate Nrf2, a
transcription factor which mediates the expression of anti-
oxidant mediators such as CAT, SOD, and HO-1 [8,11].
Notably, curcumin has been shown to act as a neuroprotectant
against methylmercury and 6-hydroxydopamine-induced neu-
rotoxicity via the up-regulation of Nrf2 and HO-1 expression
[33,34]. Similarly in the present study, we found that curcumin
may counteract colistin-induced neurotoxicity by increasing the
expression of the anti-oxidant response factors Nrf2 and HO-1
(Fig. 5;Fig.S2).
Fig. 6 Immunofluorescence
analysis of NF-κBproteinex-
pression in N2a cells. N2a cells
were pretreated with curcumin
(Cur;20μM) or bisdemethoxy-
curcumin (BDMC;20μM) for
2 h, followed by incubation with
colistin (200 μM) for an addi-
tional 24 h (n= 3). The N2a cells
were stained with anti-NF-κB
antibody. The nucleus was stained
with DAPI dye (blue). Etoposide
(Eto;50μM) was used as the
positive control treatment. The
magnified region in the colistin
only condition shows the nuclear
NF-κB. Magnification, ×40
Mol Neurobiol

To the best of our knowledge, this is the first study to report
that colistin-induced neurotoxicity involves the inflammatory
response via IL-1βand NF-κB pathway signaling with sub-
sequent induction of the inflammatory enzyme COX-2
(Fig. 5). Notably, polymyxin B has been previously reported
to induce the section of IL-1βin bone marrow dendritic cells
and macrophages and induce the expression of tumor necrosis
factor (TNF)-αin human peripheral blood mononuclear cells
[35,36]. NF-κB plays a central role in the cellular response to
diverse array of inflammatory stimuli [37]. Notably, NF-κB
activation is known to regulate the expression of over
500 genes associated with inflammation, tumorgenesis,
cellular survival/proliferation, and chemoresistance [38].
Accordingly, inhibitors of NF-κB activation such as curcumin
hold a great deal of therapeutic potential for the treatment of
inflammatory diseases. Phase I clinical trials with human sub-
jects indicated that oral curcumin doses of 8–12 g/day had no
adverse effects [39,40]. NF-κB exists in an inactive state in
the cytoplasm as a heterotrimer consisting of two subunits and
an inhibitory IκB-αsubunit [41]. NF-κB activation and nu-
clear translocation involves IKK kinase-mediated phosphory-
lation of the inhibitory IκB-αsubunit, which is released from
the NF-κB complex and is subsequently degraded [37,41,
42]. Once activated, the NF-κB subunits translocate into the
nucleus and bind to the promoters of target genes encoding
pro-inflammatory cytokines (IL-1, IL-2, IL-6, TNF-α) and
pro-inflammatory enzymes including COX-2 and inducible
nitric oxide synthase [43] which exacerbate and perpetuate
the inflammatory response by initiating catabolic processes
[44]. It is well documented that the anti-inflammatory mode
of action of curcumin involves the down-regulation of NF-kB
signaling and the mediators of inflammation (e.g., COX-2) by
inhibiting the activity of IKK kinase [42,45–47]. In the pres-
ent study, we found that curcumin can inhibit colistin-induced
expression of NF-κB and markedly decrease the expression of
p-IκB-αand COX-2; however, there was no effect on the
expression of IL-1β. It has been reported that HO-1 activation
inhibits the nuclear translocation of NF-κB[48]. Coincidently,
we observed that curcumin treatment significantly increased
the expression of HO-1 which may be partially responsible for
concomitant down-regulated expression of NF-κB.
Overall, our data suggest that curcumin attenuates colistin-
induced neurotoxicity in N2a cells by down-regulating NF-kB
and NF-kB-regulated genes involved in inflammation and ap-
optosis. These anti-inflammatory and anti-apoptotic neuropro-
tective effects are mediated partly via the down-regulation of
the phosphorylation of IκB-α, suppression of oxidative stress,
and mitochondrial-mediated apoptotic pathways (Fig. 7).
Although curcumin exhibits poor systemic availability when
administered via the oral route, various formulations for
intravenous use, including nanoparticles and liposomal
encapsulation have allowed for improved dosing and have
shown promise as anti-neoplastic agents [49–51]. Notably,
available preclinical and phase I/II data suggest that curcumin
is well tolerated, and has a good safety profile [50]. This study
provides fundamental supportive data for the clinical applica-
tion of curcumin as a co-administered neuroprotective agent
during colistin therapy of MDR Gram-negative infections.
Acknowledgements This study was supported by Key Projects in the
National Science and Technology Pillar Program during the 12th Five-
Year Plan Period (2015BAD11B03) and Chinese Universities Scientific
Fund (award number 2015DY003). T. V. is supported by a research grant
from the National Institute of Allergy and Infectious Diseases of the
National Institutes of Health (R01 AI111965). T. V. is also supported by
the Australian National Health and Medical Research Council
(NHMRC).
Compliance with Ethical Standards
Conflict of Interest The authors declare that there are no conflicts of
interest.
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