Mol Neurobiol

DOI 10.1007/s12035-017-0777-y

Tibolone Reduces Oxidative Damage and Inflammation

in Microglia Stimulated with Palmitic Acid through Mechanisms
Involving Estrogen Receptor Beta
Oscar Hidalgo-Lanussa 1 & Marco Ávila-Rodriguez 2 & Eliana Baez-Jurado 1 &
Jairo Zamudio 1 & Valentina Echeverria 3,4 & Luis Miguel Garcia-Segura 5,6 &
George E. Barreto 1

Received: 5 July 2017 / Accepted: 15 September 2017

# Springer Science+Business Media, LLC 2017

Abstract High concentrations of palmitic acid in plasma in-
crease both the inflammation associated with obesity and the
susceptibility to develop a neurodegenerative event. In the
brain, the inflammatory response is mediated by activated
microglial cells, which undergo morphological and biochem-
ical changes and can directly affect cell viability. Recent evi-
dence shows that the use of estrogenic compounds can control
microglia-induced inflammation with promising results. In
this study, we explored the actions of the synthetic steroid
tibolone on BV-2 microglia cells stimulated with palmitic ac-
id. Our results demonstrated that tibolone increased cell via-
bility and reduced nuclear fragmentation and the production
of reactive oxygen species, as well as preserved mitochondrial
membrane potential. These effects were accompanied by re-
duced nuclear translocation of NF-κB p65, upregulation of
neuroglobin, and improved antioxidant defense.
Furthermore, estrogen receptor beta (ERβ) inhibition partially
dampened tibolone’s protective actions in BV-2 cells stimu-
lated with palmitic acid. In conclusion, tibolone protects BV-2
cells by a mechanism involving ERβ and upregulation of
neuroglobin.
Keywords Palmitic acid . Obesity . Neurodegenerative
diseases . Microglia . Neuroglobin . Estrogen receptors .
Tibolone . Neuroinflammation

Electronic supplementary material The online version of this article Introduction

(https://doi.org/10.1007/s12035-017-0777-y) contains supplementary
material, which is available to authorized users.
High-fat diets significantly increase plasma-free fatty acid
* Luis Miguel Garcia-Segura
levels and are directly associated with the expression of genes
lmgs@cajal.csic.es that interfere with cell metabolism, growth, and differentiation
* George E. Barreto
[1, 2], as well as in the susceptibility to develop chronic in-
gsampaio@javeriana.edu.co; gesbarreto@gmail.com flammatory processes [3]. Recent studies have found that a
diet rich in simple sugars and saturated fatty acids is associated
1
Departamento de Nutrición y Bioquímica, Facultad de Ciencias, with decreased neurotrophic protection [4] and increased sus-
Pontificia Universidad Javeriana, Bogotá D.C., Colombia ceptibility to cognitive pathologies such as Alzheimer’s dis-
2
Departamento de Ciencias Clínicas, Facultad de Ciencias de la Salud, ease [5, 6]. Elevated levels of palmitic acid, a saturated fatty
Universidad del Tolima, Ibagué, Colombia acid, induce the expression of markers for the activation in
3
Research & Development Service, Bay Pines VA Healthcare System, glial cells and the expression of the pro-inflammatory cyto-
Bay Pines, FL 33744, USA kine TNF-α mRNA in the brain of ob/ob mice [7]. Similarly,
4
Fac. Cs de la Salud, Universidad San Sebastián, Lientur 1457, fatty acids disrupt the uptake of glucose in cells and at high
4080871 Concepción, Chile concentrations can trigger metabolic disorders, including
5
Instituto Cajal, CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain dyslipidemias, obesity, and diabetes [8]. Nowadays, obesity
6
CIBER de Investigación Biomédica en Red de Fragilidad y
has become a major public health problem and represents
Envejecimiento Saludable (CIBERFES), Instituto de Salud Carlos a common pathophysiological basis for several chronic
III, Madrid, Spain diseases [9].

The inflammatory processes underlying cerebral patholo-
gies [10, 11] may lead to a continuous and systematic deteri-
oration of the brain tissue [12–14] and activation of different
inflammation-related cells such as astrocytes and microglia
[15]. These cells may have a dual-side role, acting as protec-
tors or worsening the damage depending upon the microenvi-
ronment and type of injury [16–18]. As therapeutic ap-
proaches, the administration of neuroactive steroids such as
17ß-estradiol [19, 20], testosterone [21], and the synthetic
steroid tibolone [22] has been proven to downregulate the
inflammatory processes by acting on glial cell activation.
Previous studies have reported the action of tibolone on the
preservation of mitochondrial membrane potential [22], im-
proved cell survival, and reduced inflammation in neural cells
[23, 24]. It is noteworthy that many of these mechanisms
associated with tibolone are mediated by the activation of
estrogen receptors and by regulating the expression of survival
proteins such as Bcl-2 [25] or neuroglobin (Ngb1) [26]. Ngb1
is involved in several critical processes, such as oxygen trans-
port, the control of reactive oxygen species, and the preven-
tion of apoptosis [27–29]. Nevertheless, the effects and mech-
anisms of action of tibolone have not been explored in
microglial cells. In this study, we have addressed whether (i)
tibolone has an effect on the inflammatory response induced
by palmitic acid on microglial cells and (ii) its mechanism of
action involve the activation of estrogen receptors and/or the
expression of Ngb1.
Materials and Methods
BV-2 Cell Culture
BV-2 cell line (ATCC CRL-2469) was used as a microglial
cell model. It is a mouse cell line with macrophage morphol-
ogy derived from murine neonates and was generated by in-
fecting primary microglial cell cultures with a v-raf/v-myc
oncogenic retrovirus. BV-2 cells express non-specific esterase
activity, phagocytosis capacity, and a low peroxidase. The
BV-2 cell line has been used in numerous studies and has been
shown to share structural and functional similarities with pri-
mary microglia [30] and to retain most of the morphological,
phenotypic, and functional properties described for newly iso-
lated microglial cells [31].
Cells were maintained under exponential growth on RPMI
1640 medium (Lonza, Walkersville, USA, Cat.12-702Q),
containing 10% fetal bovine serum (Lonza, Walkersville,
USA), and penicillin 10 U/Streptomycin 10 mg/amphotericin
25 ng (Lonza, Walkersville, USA). Cultures were incubated at
37 °C in a humidified atmosphere containing 5% carbon di-
oxide. The medium was changed three times per week. Cells
were seeded in 96-well plates for death cell measurement, in
6-well plates for Western blot determinations, in 12-well
Mol Neurobiol
plates for flow cytometry determinations, and in 24-well
plates for tetramethyl rhodamine methyl ester (TMRM) and
immunofluorescence measurements.
Drug Treatment
Once the cells were seeded in multi-well plates, the culture
medium was replaced with serum-free medium in order to
decrease trophic stimulation that could affect the results of
the study. Twelve hours after palmitic acid, cells were treated
with different concentrations of tibolone (Sigma-Aldrich,
T0827); 10 nM–10 μM of 1,3,5-Tris(4-hydroxyphenyl)-4-
propyl-1H-pyrazole (PPT; ERα agonist; Sigma-Aldrich,
H6036) or 2,3-bis(4-hydroxyphenyl)propionitrile (DPN;
ERβ agonist; Sigma-Aldrich, H5915). In addition, 3 or 6 h
prior incubation with tibolone, PPT, or DPN, cell cultures
were treated with 100 nM 1,3-bis(4-hydroxyphenyl)-4-meth-
yl-5-[4-(2-piperidinyl-ethoxy)phenyl]dihydrochloride-1H-
pyrazole (MPP; ERα antagonist, Sigma-Aldrich, M7068) or
100 nM 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-
a]pyrimidin-3-yl]phenol (Phtpp; ERβ antagonist; Sigma-
Aldrich, SML1355). Tibolone, PPT, DPN, MPP, or Phtpp
were dissolved in 0.01% dimethyl sulfoxide (DMSO) as stock
solution at 5 mM, and further dilutions were made using
serum-free culture medium and without phenol red and L-
glutamine.
Palmitic Acid
We prepared a mixture containing 5% bovine serum albumin
(BSA) (Sigma, St Louis, MO, USA), 2 mM palmitic acid
(Sigma, P0500), and 2 mM carnitine (Sigma, C0283) in
RPMI with L-glutamine and serum-free conditions.
Carnitine was used to allow palmitic acid entry to the mito-
chondria. We tested the following concentrations of palmitic
acid: 100, 250, 500, 750 μM, and 1 mM for 12, 18, and 24 h.
The maximum concentration of BSA used was 2.5%, and in
all experiments, we used 2 mM carnitine. Control group
consisted in 2.5% BSA and 2 mM carnitine dissolved in
serum-free conditions.
Determination of Viability and Nuclear Fragmentation
After the experimental treatments, cells were incubated for 4 h
at 37 °C with 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium; Sigma M2128). Cells were then
lysed by the addition of DMSO. The blue formazan product
was evaluated in a plate reader at 595 nm (spectrophotometer).
The values were then normalized to the value of control
cultures without palmitate, which was considered to be
100% survival. Each trial was performed with a minimum of
three replicates per experiment.
Mol Neurobiol
Cell viability was also determined with propidium iodide
(Santa Cruz, CAS 25535-16-4); cells were stained for 15 min
with 10 μg/mL propidium iodide dissolved in PBS 1X.
Propidium iodide positive cells were quantified by fluores-
cence microscopy. Alternatively, cells were washed in PBS
and detached with trypsin (trypsin/EDTA 500:200 mg/L;
Lonza, Walkersville, USA). Propidium iodide positive cells
were then quantified by flow cytometry (Guava EasyCyte ™
Millipore cytometer, Billerica, Massachusetts, USA). Each
trial was performed with a minimum of three replicates for
each condition.
In order to confirm cell viability, we determined frag-
mentation and nuclear condensation by Hoechst 33258
staining. Cells were washed three times in PBS 1X and
fixed for 20 min in a solution of 4% paraformaldehyde at
room temperature. Subsequently, cells were washed and
labeled with Hoechst 33258 (5 mg/mL; Invitrogen) for
15 min. Cell nuclei were observed and photographed using
an inverted fluorescence microscope Olympus IX-53 (UV
excitation, filter CKX-NU N1157600) (excitation 352 nm/
emission 461 nm spectra) with an exposure time set be-
tween 80 and 100 ms to avoid the saturation of the pixels.
The number of fragmented/condensation nuclei was deter-
mined in at least eight randomly selected areas (0.03 mm2)
from each experimental group. The number of fragmented
and condensed nuclei was determined in at least eight ran-
domly selected areas of each experimental group. Data
were expressed as a percentage of fragmented and con-
densed nuclei relative to the total number of cells.
Determination of Reactive Oxygen Species
Evaluation of Superoxide Levels
Cells were treated in the dark for 30 min at 37 °C with 10 μM
dihydroethidium (DHE; Sigma, D7008) to evaluate the
superoxide radical O2−. Cells were then washed in PBS and
detached with trypsin (trypsin/EDTA 500:200 mg/L; Lonza,
Walkersville, USA) for flow cytometry analysis or maintained
on the plate to be evaluated by fluorescence microscopy. As a
positive control, we used 50 mM rotenone to induce the
production of reactive oxygen species (ROS). The
experiments were repeated in three different cultures.
Evaluation of Hydrogen Peroxide Radical
Production of hydrogen peroxide radical was evaluated by
staining with 2,7-dichlorofluorescein diacetate (DCFDA;
Sigma 35845) at a final concentration of 10 μM, in the dark
at 37 °C for 20 min. The results were quantified by flow
cytometry or fluorescence microscopy.
Determination of Mitochondrial Membrane Potential
(ΔΨm)
Mitochondrial membrane potential was assessed by
tetramethyl rhodamine methyl ester (TMRM, Sigma T5428)
staining. Cells were incubated at 37 °C for 20 min in the dark
with 500 nM TMRM. The results were quantified by flow
cytometry or fluorescence microscopy. As an experimental
control, carbonylcyanide-m-chlorophenylhydrazone (CCCP)
(Sigma-Aldrich) was used. This allowed to dissipate the mem-
brane potential and define the baseline for mitochondrial po-
tential analysis.
Determination of Mitochondrial Mass
Mitochondrial mass was evaluated by staining 10-N-nonyl
orange acridine (NAO, Invitrogen). This probe is primar-
ily used to determine non-peroxidated cardiolipin that is
present mainly in active mitochondria. Cells were stained
at 37 °C for 20 min in the dark with 200 nM NAO, after
washing twice with PBS 1X. The results were quantified
by flow cytometry or fluorescence microscopy.
Immunocytochemistry
Cells were washed in PBS 1X and fixed with 4% parafor-
maldehyde for 20 min at room temperature. Subsequently,
cells were permeabilized for 10 min in 0.1% Triton X-100
in PBS 1X. Then, cells were washed with PBS 1X, blocked
with 2% BSA + Tris-buffered saline (TBS). Subsequently,
cells were incubated overnight with the primary antibodies
for 4-hydroxynonenal (ab46545) (1:500), neuroglobin
(ab37258) (1:50), anti-NF-κB p65 (ab16502) (1:500), 3-
nitrotyrosine (ab61392) (1:500), and anti-8-
hydroxyguanosine (8-OGN) (15A3) (ab62623) (1:500).
After incubation with primary antibody, cells were washed
three times for 5 min with TBS and incubated for 45 min at
room temperature with the appropriate secondary antibody
goat anti-mouse IgG (H + L), DyLight 488 conjugate, or
goat anti-rabbit for 1 h. Finally, cells were washed with
TBS, stained with Hoechst 33258 to label the nuclei, and
photographed on an inverted Olympus IX-53 fluorescence
microscope. Quantification was performed using ImageJ
software.
Estimation of Cellular Mean Fluorescence Intensity
The calculation of mean fluorescence intensity of the cells,
for the determination of ROS, mitochondrial membrane po-
tential, and immunocytochemistry, was assessed using
ImageJ version 1.47v. The microphotographs were opened
in the software and pre-processed eliminating the back-
ground. Subsequently, 20 cells were randomly selected

using a numbered grid in each microphotograph. The mean
fluorescence value of the 20 cells was determined in 8 mi-
crophotographs for each treatment using the measure algo-
rithm of ImageJ and selecting each cell manually via ROI’s
Management. There were no variations in the conditions of
the image processing. Thus, gain of the mercury lamp, time
of exposure, and fluorochrome bleach were maintained
constant. Each assay was performed with a minimum of 6
replicate wells for each condition. The experiments were
repeated three times.
Protein Extraction and Western Blotting
BV-2 cells were lysed on ice with RIPA Lysis and
Extraction Buffer Thermo Scientific™ supplemented with
Halt™ Protease Inhibitor Cocktail, EDTA-free (100X)
(Roche). Protein content was estimated using the
Pierce™ BCA Protein Assay Kit. Equal amounts of protein
were dissolved in sample buffer containing 5% β-
mercaptoethanol and boiled. Then, proteins were separated
by electrophoresis in SDS–PAGE at 90 V for 90 min at
25 °C, transferred onto PVDF membranes for 90 min at
350 mA and 4 °C and blocked in 5% skim milk dissolved
in Tris-buffered saline containing 0.05% Tween 20 (TBS-
T), at RT for 1 h. The membranes were incubated at 4 °C
overnight with antibodies against neuroglobin (Ngb1)
(Abcam) (1:500), β-actin (Thermo Fisher) (1:3000), super-
oxide dismutase 2 (Thermo Fisher, PA5-30604) (1:1000),
Catalase (Thermo Fisher, PA5-29183) (1:1000), GPX1
(Thermo Fisher, PA5-30593) (1:1500), NF-κB p65
(abcam) (1.500), IkBα (Thermo Fisher, PA5-12186)
(1:1000), estrogen receptor beta (Thermo Fisher, PA1-
311) (1:1000), and estrogen receptor alpha (Thermo
Fisher, MA1-310) (1:1000). The immunoreactivity was vi-
sualized by incubating the membrane with specific second-
ary antibody (IRDye® Antibodies) for 1 h and detected
using Odyssey CLx Imaging System Specifications (LI-
COR Biosciences). The intensity of each band was quanti-
fied using ImageJ software (National Institutes of Health,
Bethesda, MD, USA). All data were normalized to control
values on each gel. The experiments were repeated in three
different cultures.
Neuroglobin Silencing
Cells were transfected in serum-free conditions with either
Stealth RNAi™ Ngb1 siRNA (siNgb1; Invitrogen, Carlsbad,
CA, USA) or a mismatch sequence in accordance with the
manufacturer ’s instructions, using oligofectamine
(Invitrogen) as the transfection reagent. The sequence used
for Ngb1 oligonucleotides was 5′-
CGUGAUUGAUGCUGCAGUGACCAAU-3′. The control
m i s m a t c h s e q u e n c e w a s 5 ′ -
Mol Neurobiol
UGUGAUUUAUGGUGCAGUAACCAAC-3′.
Oligofectamine and oligonucleotides (400 pM) were mixed
with Optimem. The mixture was incubated for 20 min at room
temperature, diluted with Optimem, and added to the cell me-
dium for 4 h at 37 °C. The medium was added to cells to reach
the growing conditions (i.e., 10% (v/v) serum).
Co-localization Analysis
For the analysis of the co-localization of p65 and Hoechst,
cells were incubated overnight with the primary antibody
anti-NF-κB p65 (ab16502), which was recognized using
the corresponding goat anti-rabbit secondary antibody in-
cubated for 1 h. Then, cells were co-stained with Hoechst
33258. The percentage of co-localization was determined
in for cells positive for both markers. Cells were
photographed in 4 different microscope fields accounting
for an area of 0.03 mm 2 . The co-localization plugin
Intensity Correlation Analysis 6.0 of ImageJ was used to
obtain the percentage of co-localization in each field. Four
microscope fields in 10 images for each condition were
analyzed. The results were plotted as percentage of co-
localization.
Statistical Analysis
Data obtained from this study were tested for normal dis-
tribution with the Kolmogorov–Smirnov test and for ho-
mogeneity of variances with the Levene’s test. Then, data
were examined by analysis of variance (ANOVA), follow-
ed by Dunnett’s post hoc test for comparisons between
controls and treatments and Tukey’s post hoc test for mul-
tiple comparisons between the means of treatments and
time points. Data are presented as mean ± SEM of three
independent experiments. A statistically significant differ-
ence was defined at P < 0.05.
Results
Tibolone Promoted the Survival and Preserved
the Morphology of BV-2 Cells Treated with Palmitic Acid
The analysis of time and dose-dependent curves of the ac-
tion of palmitic acid on BV-2 cells (Fig. S1) showed that
250 μM palmitic acid for 12 h reduced cell survival by
~ 50% (Fig. S1D). This was accompanied by increased
fragmented nuclei (Fig. S1E), diminished mitochondrial
membrane potential (Fig. S1F), and augmented ROS pro-
duction (Fig. S1G, H). Next, we evaluated the actions of
tibolone in our model. Cells were pre-treated or co-treated
with different concentrations of tibolone for 12, 18, or 24 h
and 250 μM palmitic acid (Fig. S2). Under these
Mol Neurobiol
Fig. 1 Effects of tibolone (Tibo) and palmitic acid (Pal) on cell viability
and nuclear fragmentation. a–b MTT and PI assays revealed that 250 μM
palmitic acid for 12 h reduced cell viability (P < 0.0001, Vh vs. Pal 12 h)
and that post-treatment with 0.01 μM tibolone for 12 h significantly
increased cell survival under palmitic acid insult (P < 0.001, Pal vs. Pal
+ Tibo 0.01 μM). c Palmitic acid increased the number of cells with
fragmented/condensed nucleus (P < 0.0001, Vh vs. Pal 12 h), and
conditions, tibolone was unable to protect from palmitic
acid. However, our results demonstrated that post-
treatment with 0.01 μM tibolone was able to preserve the
viability of cells treated with 250 μM palmitic acid (Fig. 1).
Post-treatment with 0.01 μM tibolone for 12 h significantly
increased cell viability in 25% compared to the group treat-
ed with palmitic acid alone (Fig. 1a–b, P < 0.01, Pal vs. Pal
+ Tibo 0.01 μM at 12 h). We used these parameters in next
experiments. Figure 1c shows the number of fragmented/
condensed nuclei [22, 32]. Cells treated with palmitic acid
had a significant increase by 75% in the number of
fragmented/condensed nuclei in comparison with vehicle
(P < 0.0001, Pal vs. Vh). On the contrary, tibolone reduced
by 30% the number of fragmented/condensed nuclei in
palmitic acid-treated cells (Fig. 1c, P < 0.0001, Pal vs. Pal
+ Tibo).
0.01 μM tibolone was able to attenuate the number of condensed nuclei
(P < 0.0001 Pal vs. Pal + Tibo 0.01 μM). The panel shows representative
examples of Hoechst 33258, propidium iodide (PI) stained nuclei of BV-2
cells treated with 250 μM palmitic acid for 12 h, and 0.01 μM tibolone for
12 h. Cells were photographed at magnification of ×20. Vh, vehicle. Scale
bar, 20 μm. Significant differences: ****P < 0.0001
Tibolone Preserved Mitochondrial Functions
and Reduced Oxidative Stress in BV-2 Cells Exposed
to Palmitic Acid
Palmitic acid significantly reduced in 50% the mitochondrial
membrane potential compared to vehicle (Fig. 2a, P < 0.0001,
Vh vs. Pal). Conversely, tibolone significantly increased by
40% the mitochondrial membrane potential in palmitic acid-
treated cells (Fig. 2a, P < 0.0001, Pal vs. Pal + Tibo). Similar
results were observed in NAO fluorescence. Palmitic acid
reduced mitochondrial mass in 20%, but treatment with
tibolone preserved it at levels similar to vehicle (Fig. 2b).
Regarding ROS production, we observed that palmitic acid
increased by 37% the production of superoxide (Fig. 2c,
P < 0.0001, Vh vs. Pal) and peroxide by 15% (Fig. 2d,
P < 0.001, Vh vs. Pal). On the contrary, tibolone attenuated

Fig. 2 Effects of post-treatment with 0.01 μM tibolone (Tibo) and
250 μM palmitic acid (Pal) on mitochondrial membrane potential, mito-
chondrial mass, and ROS production in BV-2 cells by flow cytometry.
Microphotographs illustrate cells stained for TMRM (Δψm, mitochon-
drial membrane potential), NAO (mitochondrial mass), and DHE (super-
oxide levels) in cells exposed to both palmitic acid and tibolone; flow
cytometry plots indicate DCFDA (peroxide levels). a Palmitic acid damp-
ened Δψm (P < 0.0001, Vh vs. Pal) and tibolone preserved mitochon-
drial potential in palmitic acid-treated cells at 12 h (P < 0.0001, Pal vs. Pal
+ Tibo). Similar effects of tibolone and palmitic acid were observed on
superoxide production by 15% (Fig. 2c, P < 0.01 Pal vs. Pal +
Tibo) and increased hydrogen peroxide by 60% (Fig. 2d,
P < 0.0001, Pal vs. Pal + Tibo) in cells under palmitic acid.
Estrogen Receptor Beta Is Involved in the Protective
Effects of Tibolone on BV-2 Cells
Previous studies have shown that estrogen receptor beta plays
a role in mitochondrial protection under different stimuli [26,
33–35]. First, we determined the protein expression of both
receptors in BV-2 cells by Western blot. Although we did not
find expression of ERα, BV-2 cells did express ERβ.
However, no significant differences were observed in ERβ
expression, regardless whether cells were subjected to
Mol Neurobiol
NAO fluorescence (b). Tibolone preserved mitochondrial mass and vol-
ume in cells upon treatment with palmitic acid (P < 0.001, Pal vs. Pal +
Tibo). The levels of superoxide ion (c) were found decreased in palmitic
acid-treated cells following administration of tibolone (P < 0.01, Pal vs.
Pal + Tibo). On the contrary, a post-treatment with tibolone augmented
the levels of hydrogen peroxide (d) in cells insulted with palmitic acid
(P < 0.0001, Pal vs. Pal + Tibo). Cells were photographed at ×20 mag-
nification. Vh, vehicle. Scale bar, 20 μm. Significant differences:
**P < 0.01, ***P < 0.001, ****P < 0.0001
tibolone or palmitic acid treatments (P > 0.05; Fig. 3a).
Likewise, no significant alterations in ERβ expression were
observed when estrogen receptor agonists were administered
(DPN and PPT, Fig. 3a). Next, we treated palmitic acid-
insulted cells with either DPN (ERβ agonist) or PPT (ERα
agonist) and assessed cell viability by PI. Our findings indi-
cated that 0.1 μM DPN was able to increase by 20% BV-2 cell
survival against palmitic acid (Fig. 3b, P ˂ 0.001, Pal vs. Pal +
DPN). On the contrary, PPT was unable to improve cell via-
bility in cells treated with palmitic acid (Fig. 3c). Finally, we
investigated whether the pharmacologic blockade of ERβ or
ERα affects the actions of tibolone, DPN, or PPT in BV-2
cells. We observed that ERβ blockade significantly dimin-
ished by 60% cell viability of microglia subjected to palmitic
Mol Neurobiol
Fig. 3 Implication of estrogen and androgen receptors in BV-2 cells. a
Western blot analysis showed that these cells do not express ERα even
under different experimental paradigms. On the contrary, ERβ expression
was detected. However, ERβ expression was not modulated when cells
were treated with vehicle (Vh), palmitic acid (Pal), tibolone (Tibo), Phtpp
(ERβ antagonist), DPN (ERβ agonist), or PPT (ERα agonist). b Post-
treatment with 0.1 μM DPN for 12 h significantly increased cell viability
in cells exposed to palmitic acid (P < 0.001, Pal vs. Pal + 0.1 μM DPN). c
Different PPT concentrations had no significant effects on cell viability in
acid and exposed to tibolone or DPN (Fig. 3d, P < 0.0001, Pal
+ Tibo vs. Phtpp + Pal + Tibo; P < 0.0001, Pal + DPN vs.
Phtpp + Pal + DPN). In contrast, blocking ERα with MPP did
not significantly affect cell viability (Fig. 3e). Finally, we also
assessed the role of androgen receptor in mediating the effect
of tibolone on BV-2 cells. Our findings showed that the an-
drogen receptor antagonist flutamide did not alter the protec-
tive effect of tibolone on palmitic acid-treated cells (Fig. 3f).
Tibolone Reduced Oxidation of Lipids, Proteins,
and Nucleic Acids in BV-2 Cells Treated with Palmitic
Acid
In this aim, we evaluated the actions of tibolone on DNA,
protein, and cell membrane oxidation in cells treated with
palmitic acid. We observed that tibolone reduced by 40%
DNA oxidation (Fig. 4a, P < 0.0001, Pal vs. Pal + Tibo), by
60% protein oxidation (Fig. 4b, P < 0.0001, Pal vs. Pal +
Tibo), and by 50% lipid peroxidation in cells treated with
palmitic acid (Fig. 4c, P < 0.0001, Pal vs. Pal + Tibo).
Similar effects on all three analyzed parameters were observed
when cells were treated with the ERβ agonist DPN.
Tibolone Regulated the Expression of Antioxidant
Enzymes
Figure S4A shows representative Western blots for each
protein under different experimental paradigms. Palmitic
cells treated with palmitic acid. d Blocking ERβ with 0.1 μM Phtpp for 3
and 6 h, in the presence of tibolone or DPN, reduced cell viability in
palmitic acid-treated cells. e Blocking ERα with MPP did not exert any
significant effect on cell viability. f Blocking androgen receptor with
different flutamide concentrations did not affect the actions of tibolone
on cell survival. Western blot lanes: 1 Vh, 2 Pal, 3 Pal + Tibo, 4 Phtpp +
Pal + Tibo, 5 Pal + DPN, 6 Phtpp + Pal + Dpn, 7 Pal + PPT. Significant
differences: **P < 0.01, ***P < 0.001, ****P < 0.0001
acid decreased by 20% the SOD expression compared
to control (Fig. S4B, P < 0.01 Vh vs. Pal). In contrast,
both tibolone and DPN increased SOD expression to
control values in palmitic acid-treated cells (Fig. S4B,
P < 0.05 Vh vs. Pal + Tibo; P < 0.01, Vh vs. Pal +
DPN). Upon ERβ blockade using Phtpp, tibolone and
DPN effects on SOD were dampened (Fig. S4B,
P < 0.01, Pal + Tibo vs. Phtpp + Pal + Tibo;
P < 0.01, Pal + DPN vs. Phtpp + Pal + DPN).
Further, we investigated the actions of tibolone and
DPN on catalase and GPX-1 expression in palmitic
acid-treated cells. Similar to SOD, palmitic acid reduced
by 10% the expression of catalase (Fig. S4C, P < 0.01,
Vh vs. Pal), but not of GPX-1 (Fig. S4D). Catalase
expression was also found decreased when cells were
treated with either tibolone or DPN in the presence of
palmitic acid (Fig. S4C, P < 0.01, Pal vs. Pal + Tibo;
P < 0.05, Pal vs. Pal + DPN). When the ERβ was
antagonized, the actions of tibolone and DPN on cata-
lase expression were blunted (Fig. S4C). GPX-1 expres-
sion was not significantly affected by palmitic acid,
tibolone, PPT, or DPN (Fig. S4D).
Tibolone and DPN Prevented the Effect of Palmitic Acid
on the Expression of Neuroglobin in BV-2 Cells
Ngb1 expression was assessed by immunocytochemistry
and Western blotting (Fig. 5). Interestingly, palmitic acid

Fig. 4 Effects of tibolone (Tibo), ERβ agonist (DPN), and ERα agonist
(PPT) on oxidative stress markers and ROS production in cells exposed to
palmitic acid (Pal). Microphotographs showed cells stained for 8OHdG,
3-NT, and 4-HNE in the presence of Vh (vehicle), Pal, DPN, or Tibo by
immunocytochemistry. a Tibolone decreased 8OHdG levels (P < 0.0001,
Pal vs. Pal + Tibo) in cells stimulated with palmitic acid, with an effect
significantly reduced by 30% neuroglobin immunofluo-
rescence in BV-2 cells in comparison to vehicle (Fig.
5a, P ˂ 0.0001, Vh vs. Pal). On the contrary, tibolone
and DPN increased by 50% Ngb1 immunoreactivity in
palmitic acid-treated cells, a level that was above to
control values (Fig. 5a, P ˂ 0.0001, Pal vs. Pal +
Tibo and Pal vs. Pal + DPN). The ERβ antagonist
Phtpp caused a significant reduction of 27 and 22% in
Ngb1 immunoreactivity in cells treated either with
tibolone (Fig. 5a, P ˂ 0.05, Pal + Tibo vs. Phtpp +
Pal + Tibo) or DPN (Fig. 5a, P ˂ 0.05, Pal + DPN
vs. Phtpp + Pal + DPN), respectively. This finding sug-
gests potential implication of ERβ in the upregulation
of Ngb1 levels by tibolone. These observations were
corroborated by Ngb1 protein expression (Fig. 5b).
Since we observed that tibolone induced the expres-
sion of Ngb1 in BV-2 cells, next we evaluated the role
played by this protein by using siRNA (Fig. S3). Ngb1
silencing did not affect the protective effect of tibolone
on the viability of cells treated with palmitic acid (Fig.
S3A). In contrast, Ngb1 silencing prevented the effect
of tibolone on superoxide ion production (Fig. S3B) and
mitochondrial membrane potential (Fig. S3C) in cells
treated with palmitic acid.
Mol Neurobiol
similar to that exerted by DPN (P < 0.0001, Pal vs. Pal + DPN). Tibolone
and DPN had similar effects by attenuating the levels of both 3-
nitrotyrosine (b), a marker of nitrated proteins, and 4-HNE (c), a marker
of lipid peroxidation (P < 0.0001, Pal vs. Pal + Tibo) under palmitic acid
exposure. Cells were photographed at ×20 magnification. Scale bar,
20 μm. Significant differences: ***P < 0.001, ****P < 0.0001
Tibolone and Palmitic Acid Modulated p65 Expression
and Nuclear Translocation
Recent studies showed that tibolone regulates the NF-κB
pathway via AKT activation [36]. Next, we investigated the
effect of palmitic acid and tibolone on p65 nuclear expression
subunit at different times using immunofluorescence (Fig. 6a).
Our findings indicated that palmitic acid induced an increase
of ~ 60% in expression of p65 at 12 h when compared to
baseline. In contrast, tibolone decreased the expression of
p65 when administered along with palmitic acid (Fig. 6a,
P < 0.0001, Pal vs. Pal + Tibo) in this same time period. Co-
localization studies using ImageJ co-localization plugin at
12 h (Fig. 6b–e) were performed and corroborated our previ-
ous findings.
The effect of tibolone, reducing p65 nuclear translocation
in cells treated with palmitic acid, was imitated by the ERβ
agonist DPN and prevented by the ERβ antagonist Phtpp
(Fig. 7a). Similar results were observed in Western blot
(WB) analysis (Fig. 7b). We investigated the expression of
IκBα in BV-2 cells. Palmitic acid significantly decreased
IκBα expression in comparison to controls (Fig. 7c,
P ˂ 0.001, Vh vs. Pal). DPN and tibolone positively modulat-
ed by 60% the IκBα expression in the cells treated with
Mol Neurobiol
Fig. 5 Expression of neuroglobin (Ngb1) in BV-2 cells stimulated with
vehicle (Vh), palmitic acid (Pal), tibolone (Tibo), ERβ antagonist
(Phtpp), and ERβ agonist (DPN) and subjected to palmitic acid.
Microphotographs showed cells stained for Ngb1 after different treat-
ments. a Immunofluorescence for Ngb1 demonstrated increased expres-
sion when cells were treated with tibolone 0.01 or 0.1 μM DPN, in the
presence of palmitic acid. On the contrary, cells treated with Phtpp
palmitic acid (Fig. 7c, P ˂ 0.01, Pal vs. Pal + Tibo and Pal vs.
Pal + DPN). The ERβ antagonist Phtpp prevented the effects
of tibolone and DPN on IκBα expression in palmitic acid-
treated cells (Fig. 7c, P ˂ 0.01, Pal + Tibo vs. Phtpp + Pal +
Tibo; P ˂ 0.01, Pal + DPN vs. Phtpp + Pal + DPN).
Discussion
Palmitic Acid Actions on BV-2 Cells
Our study for the first time characterized the effects of tibolone
on palmitic acid-treated microglial cells in vitro. Our findings
showed reduction in Ngb1 expression. b Western blot quantification of
Ngb1 under different conditions corroborated the evidences observed in
immunostaining. Cells were photographed at ×20 magnification; scale
bar, 20 μm. Western blot lanes: 1 Vh, 2 Pal, 3 Pal + Tibo, 4 Phtpp +
Pal + Tibo, 5 Pal + DPN, 6 Phtpp + Pal + DPN. Scale bar, 20 μm.
Significant differences: *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001
indicated that tibolone improved cell viability, attenuated nu-
clear condensation, reduced lipid and protein oxidation, and
preserved mitochondrial functionality. These actions were ac-
companied by a positive regulation of Ngb1 and diminished
translocation of p65 subunit of NF-κB. Our results also sug-
gest that tibolone protective effects are in part mediated by
estrogen receptor beta.
Upon increased concentration of palmitic acid, it is expect-
ed that the glycolytic and oxidative pathways result in an
imbalance of metabolic intermediates that migrate to alterna-
tive pathways to complete their metabolism. For example, a
dysregulation in the activity of serine palmitoyltransferase
may lead to an increase of dehydrosphingosine and ceramide

Fig. 6 Expression of the NF-kB p65 subunit at different times of expo-
sure to palmitic acid (Pal), vehicle (Vh), and tibolone (Tibo). a Palmitic
acid-treated cells exposed to tibolone show a reduced expression of p65 at
4 h (P < 0.0001, Pal vs. Pal + Tibo), 8 h (P < 0.0001, Pal vs. Pal + Tibo),
and 12 h (P < 0.0001, Pal vs. Pal + Tibo). Microphotographs showing p65
nuclear translocation at 12 h when cells are exposed to Vh (b), palmitic
synthesis [37, 38], which is suggested to decrease the produc-
tion of Bcl-2, an anti-apoptotic protein [39, 40]. On the other
hand, saturated fatty acids, such as palmitic acid, may impair
the mitochondrial ATP production and increase ROS produc-
tion [37, 40, 41]. Previously, Lumbertucci et al. (2008) dem-
onstrated that 100 μM palmitic acid can increase ROS pro-
duction by inducing mitochondrial electron transport chain
deterioration [16, 30, 42], inducing cytochrome C reduction
and increased production of the superoxide ion in primary
culture of rat skeletal muscle cells [43]. Indeed, Shonfeld
and Wojtczak (2008) proposed that palmitic acid may act as
a modulator of ROS production—superoxide and peroxide
species—mainly by (i) slowing down the rate of electron flow
through Complexes I, III, and IV of the respiratory chain due
to interaction within the complex subunit structure and (ii) due
Mol Neurobiol
acid alone (c), or palmitic acid and tibolone (d). e Co-localization studies
using ImageJ plugin showed the percentage of double-labeled cells for
p65 and Hoechst at 12 h. Tibolone reduced the percentage of palmitic
acid-stimulated cells positive for both p65 and Hoechst (P < 0.0001, Pal
vs. Pal + Tibo). Cells were photographed in magnification of ×20. Scale
bar, 20 μm. Significant differences: ****P < 0.0001
to their protonophoric action on the inner mitochondrial mem-
brane [44].
Tibolone Diminished the Detrimental Effect of Palmitic
Acid in BV-2 Cells
Tibolone has been described as a protective compound with
the ability to counteract detrimental effects of several dam-
aging conditions [22, 45, 46]. In our model, we observed
that BV-2 cells mainly expressed estrogen receptor beta,
suggesting a possible implication of this receptor in medi-
ating tibolone’s actions. It is reported that the activation of
estrogenic response via estrogen receptor beta is linked to
protective functions in neurons [24], astrocytes [35], and
microglia [47, 48]. It has been also shown that the
Mol Neurobiol
Fig. 7 Effect of vehicle (Vh), palmitic acid (Pal), tibolone (Tibo), ERβ
antagonist (Phtpp), and ERβ agonist (DPN) on p65 expression in cells
subjected to palmitic acid. a Immunofluorescence for p65 showed re-
duced expression when cells are treated with tibolone or DPN in the
presence of palmitic acid (P < 0.0001, Pal vs. Pal + Tibo; P < 0.0001,
Pal vs. Pal + DPN). On the contrary, upon blockade of ERβ with Phtpp,
p65 expression was found augmented. These results were corroborated
by WB, showing that tibolone and DPN attenuated p65 expression and
estrogenic response includes the activation of the PI3K-Akt
pathway [49], expression of anti-apoptotic Bcl-2 [25], and
increased expression of SOD-2 [50] and Gpx1 [51]. The
reduction in oxidative stress induced by palmitic acid in
cells treated with tibolone suggests a protective effect of
this drug on microglia. Indeed, the regulation of antioxidant
pathways and the maintenance of the mitochondrial func-
tion evidenced in our study are directly related to the
cytoprotective effect of tibolone on mitochondrial mem-
brane potential and preservation of mitochondrial function-
ality. Additionally, our results are in accordance with these
previous studies, as DPN, an ERβ agonist, modulated ROS
production and improved the mitochondrial membrane po-
tential [52–55] in BV-2 cells treated with palmitic acid.
that Phtpp was able to partially dampen this effect (b). c Quantification of
IkBα expression by WB. Although palmitic acid reduced IkBα expres-
sion in BV-2 cells, tibolone and DPN preserved its expression in cells
exposed to this fatty acid. A reduced expression of IkBα was observed
when cells are treated with Phtpp. Western blot lanes: 1 Vh, 2 Pal, 3 Pal +
Tibo, 4 Phtpp + Pal + Tibo, 5 Pal + DPN, 6 Phtpp + Pal + Dpn. Cells were
photographed in magnification of ×20. Scale bar, 20 μm. Significant
differences: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Previous evidences support the actions of estrogen recep-
tors to counteract oxidative damage [35, 56, 57]. Similar to
our study, upon blockade of ERβ, the protective effects of
tibolone and DPN were dampened, suggesting a possible im-
plication of ERβ in our model. It has been also reported that
estrogen receptor beta is localized in extranuclear compart-
ments [58], including the mitochondria [58]. For example,
Yang et al. (2009) demonstrated that knockdown of the ERβ
results in a lower resting mitochondrial membrane potential in
a model of hippocampal cells and primary hippocampal neu-
rons [58]. Similarly, ERs mediate innate immune responses
and microglial activation, as well as IL-6 production, and the
loss or ER deficiency associated with aging can lead to dereg-
ulation of the inflammatory response and increase

Fig. 8 Main effects of palmitic acid and tibolone in our model. In our
study, BV-2 microglial cells were subjected to palmitic acid stimulation.
The basal conditions of BV-2 cells included cytoplasmic p50/p65 local-
ization, preserved mitochondrial functions, and a non-reactive phenotype.
After palmitic acid stimulation, BV-2 cells undergo detrimental effects as
reduced viability, increased ROS production, and ROS-dependent cell
component degradation. Other detrimental effects included neuroglobin
downregulation and increased p50/p65 nuclear translocation.
vulnerability to ischemic injury in female mice brains [59]. It
seems like that ERβ activity is associated with metabolic and
mitochondrial functions in microglia [58, 60–63]. ERβ is lo-
cated in the cytoplasm of microglia, suggesting a protective
mechanism associated with the non-classic effects of estro-
gens, which inhibit the activation of microglia [30]. In our
results, the stimulation of BV-2 cells with palmitic acid result-
ed in a detrimental mitochondrial function, which was pre-
served following tibolone administration.
It is noteworthy that palmitic acid dampened mitochondrial
membrane potential, and that tibolone preserved mitochondri-
al function through activation of estrogen receptor beta.
Mitochondrial preservation by estrogens, upon different dam-
aging stimuli, is well documented [64–67]. Indeed, recent
studies have shown that estrogenic activity via estrogen recep-
tor beta was involved in the regulation of the protective factor
Ngb1 in astrocytes and neurons [29, 68]. Ngb1 has been
shown to be expressed in neurons, astrocytes [68], microglia
[35, 69], and the endocrine system [70]. Other reports have
detected the upregulation of Ngb1 in peri-infarct area, demon-
strating a protective reaction of the tissue against a detrimental
condition [69, 71]. Our results extend previous findings show-
ing increased Ngb1 expression in cultured astrocytes after
LPS stimulation or in an in vivo model of inflammation in-
duced by H2O2 toxicity [68, 72].
Mol Neurobiol
Interestingly, tibolone was able to counteract the detrimental effects of
palmitic acid by eliciting an increased viability and diminishing ROS
production. Tibolone also modulated the levels of key antioxidant pro-
teins such as SOD and catalase. More importantly, tibolone upregulated
neuroglobin in palmitic acid-treated cells, and upon ERβ blockade,
neuroglobin expression was found reduced and this partially attenuated
tibolone’s protective action on BV-2 cells
Role of Tibolone in the Inflammatory Response
in the Model
It is important to point out that microglia have the ability to
induce an inflammatory response that includes the release of
cytokines and chemokines, and activation of NF-κB down-
stream pathway [57, 73]. Other inflammatory mediators in-
clude growth factors, apoptotic modulators, NLRP3, and
Aim2 [74–76]. Free fatty acids can increase the permeability
of the blood–brain barrier allowing the infiltration and activa-
tion of inflammatory modulators. These modulators can reg-
ulate the activation of microglia by conditioning their re-
sponse and altering metabolic processes in the brain, thus
increasing the oxidative damage [75, 77]. The inflammatory
response can be modulated by various factors or molecules,
including steroid hormones such as estradiol or SERMs [78].
Several studies have confirmed the modulation of the immune
response by steroid metabolites as estrogen and progesterone
[14, 69, 78–80].
Since some secreted pro-inflammatory factors are under
control of upstream NF-κB pathway, a critical signal transduc-
tion pathway that regulates pro-inflammatory responses [30,
57], we determined the response of NF-κB signaling transduc-
tion by palmitic acid and tibolone. Our results demonstrated
that NF-κB p65 nuclear translocation was stimulated by
Mol Neurobiol
palmitic acid insult, and this action was reversed by tibolone
administration, which is in agreement with previous studies
[81–83]. We demonstrated that palmitic acid induced a detri-
mental oxidative stress that involved the NF-κB pathway. This
result is in accordance with a previous study suggesting that
ROS and oxidative damage induce NF-κB activation [76].
The estrogenic action of tibolone associated with ERβ reduces
the expression of inflammatory factors [26], as p65 expression
was also modulated by the activation of ERβ. In our study,
blocking ERβ increased the expression of p65, thus notably
inhibiting the action of tibolone (Fig. 8). On the other hand,
we observed that DPN and tibolone induced similar actions on
p65 expression, and upon blocking ERβ, their action was
found reduced. These results are supported by Frasor and
colleagues (2009), who described an association between
ERs and NF-κB suggesting a co-repression activity [73].
Our findings also demonstrated that palmitic acid increased
the expression of p65 subunit and decreased the expression of
IkBα, an upstream regulator of NF-κB, suggesting a possible
pro-inflammatory preconditioning of palmitic acid in BV-2
cells as indicated by Acton (2012) in an astrocytic model
stimulated with LPS [84]. Interestingly, all the pro-
inflammatory activity was reversed in part by tibolone admin-
istration, suggesting a novel role of tibolone in modulating the
inflammatory response via ERβ. Finally, Biswas et al. (2005)
described that while anti-inflammatory effects of ER activa-
tion have been observed, a non-classical and non-genomic
effect of estrogen receptors has also been elucidated. This
includes the activation of NF-κB and its inflammatory down-
stream response, suggesting that molecules that bind ERs and
selectively block NF-κB activation may have a potential ther-
apeutic effect [85].
In conclusion, our results demonstrated the protective ef-
fects of tibolone in BV-2 cells against palmitic acid-induced
oxidative stress and inflammation. Tibolone activates estro-
genic activity via ERβ and modulates the expression of
Ngb1 and NF-κB components in order to reduce mitochon-
drial damage and inflammation. However, further experi-
ments are needed to confirm the involvement of tibolone in
regulating other inflammatory mediators on in vitro and
in vivo models.
Acknowledgments We acknowledge support from Ministerio de
Economía y Competitividad (MINECO; grant number BFU2014-
51836-C2-1-R), from CIBERFES, and Fondos FEDER.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
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