Neurobiology of Disease 140 (2020) 104814

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Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi

Neuroprotection by dihydrotestosterone in LPS-induced neuroinflammation T

b,1 a,b,1 b b b b,1,⁎
Lei Yang , Renyuan Zhou , Yu Tong , Pengfei Chen , Yu Shen , Shuai Miao ,
⁎
Xiaoqiang Liua,1,
a
Department of Urology, Tianjin Medical University General Hospital, Tianjin Medical University 300070 Tianjin, China
b
Department of Urology, Jing'an District Central Hospital, Fudan University, Shanghai 200040, China

A R T I C LE I N FO A B S T R A C T

Keywords: Microglia-induced neuroinflammation plays a vital role in the etiology and progression of neurodegenerative
Lipopolysaccharide diseases, including Alzheimer's disease, Parkinson's disease and multiple sclerosis. The neuroprotective role of
Neuroinflammation androgens, including testosterone and its metabolite dihydrotestosterone (DHT), has been increasingly de-
Dihydrotestosterone monstrated in these diseases, but few studies investigated the effects of androgen on neuroinflammation. This
Cognitive impairment
study investigated the role of DHT in lipopolysaccharide (LPS)-induced neuroinflammation, neuronal damage
Sickness behavior
and behavioral dysfunction, as well as underlying mechanisms. We showed that DHT inhibited LPS-induced
release of proinflammatory factors, including TNF-α, IL-1β, IL-6; iNOS, COX-2, NO, and PGE2 in BV2 cells and
primary microglia by suppressing the TLR4-mediated NF-κB and MAPK p38 signaling pathways, thus protecting
SH-SY5Y neurons from inflammatory damage induced by activated microglia. In an LPS-induced neuroin-
flammation mouse model, endogenous DHT depletion by castration exacerbated inflammatory responses by
upregulating the levels of TNF-α, IL-1β, IL-6, iNOS, and COX-2 in the serum and brain by increasing the LR4-
mediated NF-κB and MAPK pathway activation, but these effects were restored by exogenous DHT supple-
mentation. Moreover, DHT also regulated the mRNA levels of the anti-inflammatory cytokines IL-10 and IL-13 in
the brain. In addition, DHT modulated the expression of Aβ, the apoptotic proteins caspase-3, Bcl-2, and Bax,
and synaptophysin, as well as neuronal damage in LPS-treated mouse brains. Further behavioral tests revealed
that DHT ameliorated LPS-induced spatial and learning impairment and motor incoordination, and partly im-
proved the locomotor activity in LPS-injected mice. Therefore, this study suggests that DHT exerts anti-neu-
roinflammatory and neuroprotective effects; thus, androgen replacement therapy is a potential therapeutic
strategy for improving cognitive and behavioral function in neuroinflammation-related diseases.

1. Introduction alleviation of neuroinflammation-related diseases.

Neuroinflammation plays a vital role in the etiology and progression
of neurodegenerative diseases, including Alzheimer's disease (AD),
Parkinson's disease (PD), and multiple sclerosis (MS) (Glass et al.,
2010). As resident macrophages in the brain, microglia can be activated
and trigger the innate immune response by sensing exogenous neuro-
toxic substances and proinflammatory stimuli. Activated microglia lead
to neuroinflammatory responses, neuronal damage and cognitive dys-
function (Graeber et al., 2011; Liu and Hong, 2003). Thus, the reg-
ulation of neuroinflammation represents a potential strategy for the
Lipopolysaccharide (LPS) is the polysaccharide component of gram-
negative bacteria that binds to Toll-like receptor 4 (TLR4) in microglia,
eliciting an innate immune response (Lehnardt et al., 2003). The in-
teraction between LPS and TLR4 activates TLR4-mediated nuclear
factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK)
signaling pathways, including P38, AKT, ERK and JNK, thus triggering
the release of proinflammatory cytokines, including tumor necrosis
factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) (He et al.,
2018; Lu et al., 2008). The proinflammatory mediators nitric oxide
synthase (iNOS) and cyclooxygenase-2 (COX-2) are also upregulated

Abbreviations: AD, Alzheimer's disease; Aβ, amyloid-β; CCK-8, Cell Counting kit-8; COX-2, cyclooxygenase-2; DHT, dihydrotestosterone; ELISA, Enzyme-linked
immunosorbent assay; iNOS, inducible NO synthase; IL, interleukin; LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; MAPK, mitogen-activated protein kinase;
NO, nitric oxide; NF-κB, nuclear factor-kappa B; OFT, Open field test; PD, Parkinson's disease; PT, Pole test; RT-PCR, Reverse transcription-polymerase chain reaction;
PGE2, prostaglandin E2; MWM, Morris Water Maze; MS, multiple sclerosis; TNF-α, tumor necrosis factor-α
⁎
Corresponding authors.
E-mail addresses: msdiyi@163.com (S. Miao), 15233367861@163.com (X. Liu).
1
The first two authors (Lei Yang & Renyuan Zhou) contributed equally to this work.

https://doi.org/10.1016/j.nbd.2020.104814
Received 19 December 2019; Received in revised form 16 February 2020; Accepted 18 February 2020
Available online 19 February 2020
0969-9961/ © 2020 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
L. Yang, et al.
and induce the production of nitric oxide (NO) and prostaglandin E2
(PGE2), which further promote neuroinflammation (Zhou et al., 2020).
Numerous studies have shown that systemic injection of LPS is a potent
activator of neuroinflammation, resulting in neurodegeneration and
abnormal cognition and behavior (Huffman et al., 2019; Qin et al.,
2007; Vasconcelos et al., 2014).
Generally, the incidence of AD is higher and the symptoms worsen
faster in women than in men (Oveisgharan et al., 2018). The sex dif-
ference in incidence of AD is regarded mainly as estrogen dependent
since estrogen dramatically decreases after menopause (Merlo et al.,
2017). Therefore, numerous studies have focused on the protective ef-
fect of estrogen in AD (Villa et al., 2016), with less emphasis on an-
drogen. The neuroprotective role of androgens, including testosterone
and its metabolite dihydrotestosterone (DHT), has been investigated in
preclinical settings, such as in AD, PD and MS models, wherein an-
drogens have been proven to exert anti-apoptotic and antioxidative
stress functions, reduce AD-related neuropathology, and improve cog-
nitive performance (Giatti et al., 2015; Khasnavis et al., 2013; Rosario
et al., 2006). However, few studies investigate the effects of androgen
on neuroinflammation. The anti-inflammatory properties of androgens
in peripheral inflammatory diseases have been broadly investigated in
animal models and clinical studies (Mohamad et al., 2019; Traish et al.,
2018). However, the effects of androgens on cognitive function in
clinical studies are still a matter of debate. On the one hand, age-related
androgen depletion increases the risk of neurodegenerative diseases
(Okun et al., 2004; Seidl and Massman, 2015); also, prostate cancer
patients with androgen deprivation therapy are more susceptible to
cognitive impairment (Nead et al., 2016, 2017), suggesting that an-
drogens protect against cognitive decline. On the other hand, supple-
mentation of androgens fails to improve cognitive function (Resnick
et al., 2017). Thus, we hypothesized that the pre-existing pathological
condition of subjects affects the efficacy of androgen treatment.
The aim of the present study was to analyze the protective role of
DHT in neuroinflammatory conditions induced by LPS in vitro and in
vivo, including the role of DHT in LPS induced microglial activation,
neuronal damage and cognitive and behavior abnormalities, as well as
the underlying mechanism.
2. Materials and methods
2.1. Cell culture and treatment
The BV2 microglial cell lines and SH-SY5Y neurons were cultured in
Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented
with 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin, and
100 μg/ml streptomycin (Gibco); and incubated at 37 °C in a humidified
incubator containing 95% air and 5% CO2. Primary microglial cultures
were prepared as described previously (Saura et al., 2003). Briefly,
whole brains of C57BL/6 mice, aged 1–2 days, devoid of meninges and
blood vessels, were mechanically dissociated and disrupted using a 70-
μm nylon mesh. The isolated cells were cultured for 14 days in DMEM/
F12 (HyClone) supplemented with 10% FBS, 100 U/ml penicillin, and
100 μg/ml streptomycin. Then the mixed primary glial cells were in-
cubated with trypsin solution (0.25% trypsin, and 1 mM EDTA in
Hank's balanced salt solution) diluted 1:4 in DMEM/F12. After the
primary astrocyte layer was fully detached, DMEM/F12 containing 10%
FBS was added, the supernatant was aspirated, and the remaining pri-
mary microglia were used for experiments when the cells reached 95%
purity (Fig. S1).
DHT (MedChemExpress, Shanghai, China) and the TLR4 inhibitor,
CLI-095 (Fushen, Shanghai, China) were dissolved in dimethyl sulf-
oxide (DMSO; Sigma-Aldrich, USA), and the final DMSO concentration
was < 0.1%. The cells were pretreated with DHT (10 nM) for 24 h with
or without CLI-095 (1 μM) for 30 min followed by incubation with
100 ng/ml LPS (O26:B6, Sigma-Aldrich) for the indicated times
(45 min, 6 h, and 24 h). The concentrations of DHT, LPS and CLI-095
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Neurobiology of Disease 140 (2020) 104814
were chosen according to previously described studies (Kim et al.,
2019a,b; Yao et al., 2017).
2.2. Animal surgery and LPS injection
Male C57BL/6J mice, aged 4–6 weeks (18–22 g), were purchased
from Vital River Laboratory Animal Center (Beijing, China) and main-
tained under standard laboratory conditions. The mice were randomly
housed in cages with a 12 h light/dark cycle, at 40% humidity and a
temperature of 24 ± 1 °C, and had a free access to water and food. The
study was approved by the Institutional Animal Care and Use
Committee of Fudan University, of Shanghai Medical College.
The mice were gonadectomized or sham gonadectomized under
general anesthesia, efforts were made to minimize suffering. Mice un-
derwent bilateral orchiectomies by removing the testes, epididymis and
epididymal fat. The sham-operated mice received the same surgical
operation except that the exposed testes were not removed. Castrated
mice were given 15 days to ensure depletion of endogenous androgen
prior to sequent experiments.
The mice were randomly divided into five groups (n = 10–15 mice/
group): the control group, castration group, LPS group,
castration + LPS group, and castration + DHT + LPS group (dihy-
drotestosterone, 5 mg/kg, s.c.). DHT was administered once daily for 15
consecutive days after castration for two weeks. The doses of DHT were
chosen based on earlier reports (Giatti et al., 2015). According to pre-
vious studies (Huffman et al., 2019; Vasconcelos et al., 2014), LPS
(1 mg/kg body weight) was administered intraperitoneally once a day
for 5 days to induce a chronic neuroinflammatory model. A single dose
of LPS (5 mg/kg body weight) was used to induce acute neuroin-
flammation (Oliveira-Lima et al., 2019).
2.3. Cell viability assay
BV2 cells, primary mouse microglial cells and SH-SY5Y neurons
were seeded in 96-well culture plates at a density of 1 × 104 cells/well.
Cell viability was evaluated at 24 h after treatment with the indicated
drugs or conditioned medium by a Cell Counting kit-8 assay (CCK-8,
Beyotime, China). A volume of 10 μl CCK-8 dye was added to each well,
and then the plate was incubated for another 2 h in a humidified in-
cubator at 37 °C. The optical density of each well was measured at a
wavelength of 450 nm.
2.4. Enzyme-linked immunosorbent assay (ELISA)
The levels of TNF-α, IL-1β, IL-6, and PGE2 ((Novus Biological,
Littleton, CO, USA)) in the cell culture medium, brain tissue homo-
genate and serum were measured with ELISA kits following the man-
ufacturer's recommendations.
2.5. Griess assay
The nitric oxide (NO) level in the culture medium was determined
by measuring nitrite, a stable NO catabolite, using the Griess reagent
(Beyotime, China). BV2 microglial cells (1 × 105 cells/ml) were sti-
mulated in 24-well plates with or without 10 nM DHT for 24 h prior to
LPS (100 ng/ml) treatment for 24 h, and then 50 μl of each culture
supernatant was mixed with an equal volume of Griess reagent at room
temperature for 10 min. The absorbance was measured at a wavelength
of 540 nm using an ELISA microplate reader, and the level of nitrite was
calculated based on a standard sodium nitrite curve.
2.6. Immunocytochemistry
BV2 cells and primary microglia were washed three times with 1×
PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized and
blocked with 0.2% Triton X-100 and 10% goat serum in PBS for 1 h.
L. Yang, et al. Neurobiology of Disease 140 (2020) 104814

Fig. 1. DHT decreased LPS-induced proinflammatory factor expression in BV2 microglial cells. BV2 cells were pretreated for 24 h with 10 nM DHT followed by
100 ng/ml LPS for 24 h (a–c) and 6 h (d–h); a) iNOS levels were detected by Western blotting (n = 3). Protein band intensity was normalized to β-actin and is
expressed as the fold difference relative to the control group. NO (b, n = 3) and TNF-α (c, n = 3) levels in the culture supernatant were measured by the Griess assay
and ELISA, respectively. e–h (n = 3) TNF-α, IL-1β, IL-6, iNOS and COX-2 transcription levels were analyzed by quantitative real-time PCR and normalized to that of
β-actin. The data represent the three independent experiments. ⁎⁎⁎P < .001 vs. the control group; #p < .05, ##p < .01 and ###p < .001 vs. the LPS group; One-
way ANOVA analysis of variance.

The glass slides were mounted and incubated with rabbit-anti-Iba-1
(1:500) or anti-GFAP (1:500) overnight at 4 °C in a humidified
chamber. The next day, the cells were washed three times with 1 × PBS
and incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 488,
1:1000) for 1 h and the nuclei were stained with DAPI for 15 min.
Images were captured using a fluorescence microscope (Leica) and
analyzed using ImageJ software.
2.7. Immunohistochemistry and Nissl staining
For immunohistochemistry, brain tissues were fixed with 4% par-
aformaldehyde and then permeabilized for 1 h in PBS with 0.2% Triton
X 100 and 10% BSA at room temperature. Then, the frozen sections
were incubated with primary antibodies at 4 °C overnight. The fol-
lowing day, the tissues were washed three times with 1× PBS. Then,
the sections were incubated in 3% H2O2 to eliminate endogenous per-
oxidase activity. Following three-time washes in 1× PBS, the sections
were incubated with horseradish peroxidase (HRP)-conjugated poly-
clonal rabbit Ig G (Cell Signaling Technology, Danvers, MA, USA) at
37 °C for 1 h. The sections were developed and stained with
diaminobenzidine (Absin Biological, Shanghai, China). After dehydra-
tion and transparentizing, the sections were observed under a light
microscope and quantified by Image-Pro Plus 6.
For Nissl staining, the brain sections were washed three times with
0.01 M PBS for 15 min, followed by staining with a 0.5% cresyl violet
solution for 10 min. Then, the sections were rinsed with distilled water,
and dehydrated in in a graded series of alcohol (70, 95, and 100%).
Tissue slices were transparentized with xylene for 5 min, and covered
with cover slips by using neutral balsam (non-florescent). Next, the
slices were observed and photographed under a microscope. The
numbers of normal neurons were quantified by Image-Pro Plus 6 soft-
ware.
2.8. Western blot analysis
The cells and the brain tissue were lysed, and the protein con-
centration was quantified by a BCA protein assay kit (Beyotime, China)
according to the manufacturer's instructions. A quantity of 20–40 μg of
total proteins was separated by 8–10% SDS-PAGE, and and transferred
to polyvinylidene difluoride membranes and incubated with the

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L. Yang, et al. Neurobiology of Disease 140 (2020) 104814

Fig. 2. DHT inhibited the LPS-induced TLR4/NF-κB signaling pathway in BV2 cells. BV2 cells were pretreated for 24 h with 10 nM DHT followed by 100 ng/ml
LPS for 45 min (a–b, d) and 24 h (c, e–f); a)–b), d)–e) TLR4, MyD88, p-P65, P65 and iNOS protein levels were detected by Western blotting (n = 3). Protein band
intensity was normalized to β-actin and is expressed as the fold difference relative to the control group. TNF-α (c, n = 3) and NO (f, n = 3) levels in the culture
supernatant were measured by the ELISA and Griess assay. The data represent the three independent experiments. ⁎⁎P < .01 and ⁎⁎⁎P < .001 vs. the control group;
##
p < .01 and ###p < .001 vs. the LPS group; $$p < .01 and $$$p < .001 vs. CLI-095 + DHT group; One-way ANOVA analysis of variance.

primary antibodies (Table S1) at 4 °C overnight. After incubation with
horseradish peroxidase-conjugated goat/mouse anti-rabbit IgG
(1:1000) for 1 h at room temperature, the membranes were visualized
by enhanced chemiluminescence (Affinity, China) scanned by an ima-
ging system and then quantified using ImageJ software.
2.9. Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA from cells and brain tissues was extracted using TRIzol
reagent (Beyotime, China) according to the manufacturer's instructions.
Total RNA was reverse transcribed into cDNA using Hifair ™ II 1st
strand cDNA synthesis SuperMix kit (YeaSen, Shanghai, China), and RT-
PCR was performed using SuperReal PreMix Plus (TianGen, Beijing,
China) according to the manufacturer's instructions. cDNA was subse-
quently amplified by PCR with specific primers (Table S2). The Ct va-
lues were normalized to β-actin, and the data were analyzed by using
the 2-ΔΔCt method.
2.10. Behavioral test
2.10.1. Morris water maze (MWM)
The MWM test was conducted as described previously (Zhao et al.,
2019) to evaluate the memory and learning performance. Using video
tracking software, behavioral tests were recorded daily at 6 h after drug
injection. The test was performed three times a day for 5 days during
the training period (Days 21–25), with three randomized starting
points. Each test lasted for 1 min or ended as soon as the mouse reached
the submerged platform. After two days of rest (Day 26–27), latency
was measured to assess the time taken to reach the hidden platform on
4 consecutive days (Days 28–32). To evaluate memory consolidation, a
probe test was conducted 24 h after the water maze test (Day 33) by
removing the platform and allowing the mice to swim freely for 1 min.
Consolidated spatial memory was evaluated by analyzing the time spent
in the target quadrant area and the crossing numbers and time over the
previously hidden platform.
2.10.2. Open field test (OFT)
The OFT was performed to evaluate the locomotor activity of the
mice as previously described (Oliveira-Lima et al., 2019). The mice
were individually placed into the center of an open field apparatus
(30 × 30 × 60 cm) with nine virtual quadrants (10 × 10 cm each). At
24 h after drug administration, each mouse was permitted to freely
explore for 6 min including 2 min of adaptation to the apparatus and
4 min of testing. The number of crossing, grooming, rearing and the
total distance were analyzed by blinded observers to reduce bias. The
device was cleaned with 5% alcohol to avoid odor clues after each test.

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L. Yang, et al. Neurobiology of Disease 140 (2020) 104814

Fig. 3. DHT reduced LPS-induced proinflammatory factor expression in primary microglia. Primary microglia were pretreated for 24 h with 10 nM DHT
followed by 100 ng/ml LPS for 24 h (a–c) and 6 h (d–h); a) Cox-2 levels were detected by Western blotting (n = 3). Protein band intensity was normalized to β-actin
and is expressed as the fold difference relative to the control group. PGE2 (b, n = 3) and IL-1β (c, n = 3) levels in the culture supernatant were measured by ELISA.
e–h (n = 3) TNF-α, IL-1β, IL-6, iNOS and COX-2 transcription levels were analyzed by quantitative real-time PCR and normalized to that of β-actin. The data
represent the three independent experiments. ⁎⁎P < .01 and ⁎⁎⁎P < .001 vs. the control group; #p < .05, ##p < .01 and ###p < .001 vs. the LPS group; One-way
ANOVA analysis of variance.

2.10.3. Pole test (PT)
The PT was conducted to measure the motor coordination of the
mice as previously reported (Kim et al., 2019a,b). Briefly, the mice were
subjected to two days of training before testing. The test was performed
at 24 h after drug treatment. The following three times were recorded:
the time it took for the mouse to swivel from the upper half to the lower
half, the time it took for the mouse to climb down from the upper half to
the bottom of the lower half, and the time it took for the mouse to
complete the two steps above.
2.11. Statistical analysis
The statistical analysis was performed with one-way ANOVA fol-
lowed by Tukey's test. A p value < .05 was considered statistically
significant. All figures were generated by using GraphPad Prism 5
software.
3. Results
3.1. DHT inhibited the LPS-induced proinflammatory response in BV2 cells
and primary microglia
By The CCK-8 assay, results showed that 10 nM DHT had no effect
on the viability of BV2 cells and primary microglia with or without
100 ng/ml LPS treatment; moreover, this concentration restored LPS-
induced cell morphological changes (Fig. S.1), indicating that 10 nM
DHT inhibited microglial activation, as previously reported (Yao et al.,
2017). Therefore, we further determine whether pretreatment with
DHT reduced proinflammatory cytokines and mediators in LPS-stimu-
lated microglial cells. We showed that DHT significantly reduced LPS-
induced iNOS, NO and TNF-α expression levels in BV2 cells (Fig. 1a-c),
and decreased the expression levels of COX-2, PGE2 and IL-1β in LPS-
stimulated primary microglia (Fig. 3a–c). Consistently, we also ob-
served that DHT significantly reduced LPS-induced TNF-α, IL-1β, IL-6,
iNOS and COX-2 mRNA levels in BV2 cells (Fig. 1d–h) and primary
microglia (Fig. 3d–h). These data suggest that DHT exerts an anti-in-
flammatory effect in activated microglia.
3.2. DHT suppressed the TLR4-mediated NF-κB and MAPK p38 signaling
pathways in LPS-treated BV2 cells and primary microglia
The microglial proinflammatory response is attributed to the acti-
vation of TLR4 by LPS, which triggers downstream inflammatory sig-
naling pathways, including NF-κB and MAPK, leading to protein
phosphorylation and nuclear translocation, and ultimately to the pro-
duction of proinflammatory cytokines and mediators (Lehnardt et al.,

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L. Yang, et al. Neurobiology of Disease 140 (2020) 104814

Fig. 4. DHT inhibited the LPS-induced TLR4/MAPK p38 signaling pathway in primary microglia. Primary microglia were pretreated for 24 h with 10 nM DHT
followed by 100 ng/ml LPS for 45 min (a–b) and 24 h (c–e); a)–c) TLR4, p-P38, P38 and Cox-2 protein levels were detected by Western bloting (n = 3). Protein band
intensity was normalized to β-actin and is expressed as the fold difference relative to the control group. IL-1β (d, n = 3) and PGE2 (e, n = 3) levels in the culture
supernatant were measured by ELISA. The data represent the three independent experiments. ⁎⁎P < .01 and ⁎⁎⁎P < .001 vs. the control group; #p < .05,
##
p < .01 and ###p < .001 vs. the LPS group; $p < .05 vs. the CLI-095 + DHT group; One-way ANOVA analysis of variance.

Fig. 5. DHT protected SH-SY5Y neurons from neurotoxicity induced by activated microglia. a) SH-SY5Y cells were treated with 10 nM DHT and/or 100 ng/ml
LPS for 24 h and b) were incubated for 24 h with conditioned medium from cultures of BV2 cells and primary microglia. Cell viability was assessed by CCK8 assay
(n = 3). The data represent the three independent experiments. ⁎⁎P < .01 and ⁎⁎⁎P < .001 vs. the control group; ##p < .01 vs. the LPS group; NS: no significance;
One-way ANOVA analysis of variance.

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L. Yang, et al. Neurobiology of Disease 140 (2020) 104814

Fig. 6. Effects of DHT on LPS-induced activation of microglia and astrocytes in the mouse brain. Wild-type C57BL/6 mice were injected with DHT (5 mg/kg,
s.c.) or oil daily for 15 days, followed by injection with LPS (5 mg/kg, i.p.) or saline. a) Immunohistochemistry was conducted with anti-Iba-1 and anti-GFAP
antibodies 24 h after LPS injection (n = 4–5). b) Iba-1 and GFAP protein level was detected by Western bloting (n = 4). Protein band intensity was normalized to β-
actin. Quantification of the data is expressed as the fold difference relative to the control group. *P < .05 and *** P < .001 vs. the control group; #P < .05,
##
P < .01 and ###P < .001 vs. the LPS group; $P < .05, $$P < .01 and P$$$ < .001 vs. the Castration + LPS group; One-way ANOVA analysis of variance.

2003). By evaluating the effects of DHT on these signaling pathways in
microglia, we found that DHT suppressed LPS-induced increases in the
protein levels of TLR4, and MyD88 and phospho-NF-κB p65 (p-P65) in
BV2 cells (Fig. 2a–b), as well as the protein levels of TLR4, and
phospho-MAPK p38 (p-P38) in primary microglia (Fig. 4a). To further
determine whether TLR4-mediated NF-κB and MAPK signaling was
required for DHT-mediated anti-inflammatory effects, we assessed the
effect of CLI-095, a TLR4 inhibitor, on both LPS and DHT-treated mi-
croglia as previously described (Kim et al., 2019a,b). The results
showed that CLI-095 and DHT had similar inhibitory effects on the
expression of TNF-α, p-P65, iNOS and NO in LPS-stimulated BV2 cells
(Fig. 2c–f), and the expression of p-P38, COX-2, IL-1β and PGE2 in LPS-
stimulated primary microglia (Fig. 2b–e); furthermore, DHT combined
with CLI-095 further inhibited the expression of these proinflammatory
mediators compared with that of the CLI-095 + LPS group, suggesting
that DHT suppressed NF-κB and MAPK activation in microglia mainly
through downregulation of TLR4 expression.
3.3. DHT protected SH-SY5Y neurons from activated microglia-induced
neurotoxicity
LPS-stimulated microglial activation damages neurons. Thus, we
investigated the neuroprotective effects of DHT in neurons exposed to
toxic effects of activated microglia. Our findings revealed that condi-
tioned medium from LPS-treated BV2 cells and primary microglia cul-
tures decreased the viability of SH-SY5Y cells, while the conditioned
medium from microglial cultures were treated with DHT prior to LPS
treatment increased SH-SY5Y cell viability (Fig. 5b–c). Moreover, the
same concentration of DHT and LPS as in the microglial supernatants
had no effect on the viability of SH-SY5Y cells (Fig. 5a). These results
indicate that proinflammatory cytokines released into the culture
medium by microglia upon LPS stimulation decrease the viability of SH-
SY5Y cells, while DHT pretreatment counters these effects.

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L. Yang, et al. Neurobiology of Disease 140 (2020) 104814

Fig. 7. Effects of DHT on LPS-induced systemic and brain inflammation. a)–f) The proinflammatory cytokine TNF-α, IL-1β and IL-6 in the serum and brain were
measured by ELISA (n = 5); g)–i) Transcriptional levels of anti-inflammatory cytokines IL-4, IL-10 and IL-13 were analyzed by quantitative real-time PCR (n = 4) 3 h
after LPS injection. j) iNOS and Cox-2 protein levels in the brain was detected by Western blotting (n = 4) 24 h after LPS injection. *P < .05, *P < .01 and ***
P < .001 vs. the control group; #P < .05, ##P < .01 and ###P < .01 vs. the LPS group; $P < .05, $$P < .01 and $$$P < .001 vs. the Castration + LPS group;
One-way ANOVA analysis of variance.

3.4. Effect of DHT on the activation of microglia and astrocytes in LPS-
injected mouse brains
According to the above in vitro findings, we examined whether DHT
regulated LPS-induced activation of microglia and astrocytes in vivo.
LPS-injected mice showed a significant increase in the number of GFAP-
and Iba1-reactive cells, whereas castration markedly increased these
cells in the cortex and hippocampus. Interestingly, DHT supplementa-
tion in castrated mice restored microglial and astrocyte activation
(Fig. 6a). Consistent with the immunohistochemical results, Western
blot analysis also showed that DHT regulated Iba-1 and GFAP protein
levels in LPS-injected mouse brains (Fig. 6b). These data indicate that
DHT modulates microglial and astrocyte activation in LPS-induced
neuroinflammation.
3.5. Effects of DHT on LPS-induced systemic and brain inflammation
Systemic LPS injection affects inflammatory cytokine production in
the serum and brain (Oliveira-Lima et al., 2019). We investigated the
effect of DHT on the inflammatory reaction in LPS-stimulated mice. We
observed that LPS induced a significant increase in TNF-α, IL-1β and IL-
6 in the serum (Fig. 7a–c) and brain homogenates (Fig. 7d–f); castration
substantially amplified the LPS-induced release of these cytokines, but
the cytokine levels were dramatically restored by the administration of
DHT. Similarly, DHT also modulated the mRNA expression of the anti-
inflammatory cytokines IL-10, and IL-13, but failed to affect IL-4 tran-
scription (Fig. 7g–i). Moreover, the protein expression of iNOS and
COX-2 was increased in LPS-injected mice, and DHT administration
abrogated the further increase in these proteins in the cortex and

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Fig. 8. Effects of DHT on the TLR4-mediated NF-κB and MAPK pathways in LPS-injected mouse brains. a)–g) TLR4, NF-κB (p-P65 and P65) and MAPK (p-P38,
P38, p-AKT, AKT, p-ERK, ERK, p-JNK, and JNK) protein levels in the brain was detected by Western blotting (n = 4) 24 h after LPS injection. Protein band intensity
was normalized to β-actin and is expressed as the fold difference relative to the control group. **P < .01 and *** P < .001 vs. the control group; #P < .05 and
##
P < .01 vs. the LPS group; $P < .05 and $$P < .01 vs. the Castration + LPS group; NS: no significance; One-way ANOVA analysis of variance.

hippocampus of LPS-treated castrated mice (Fig. 7j). These data suggest
that DHT regulates the LPS-mediated inflammatory response systemi-
cally and in the brain.
3.6. Effects of DHT on TLR4-mediated NF-κB and MAPK pathway
activation in LPS-injected mouse brains
The in vitro findings revealed that DHT inhibited the TLR4-medi-
ated NF-κB and MAPK pathways in LPS-treated microglia. Thus, we
further investigated whether DHT regulated the activation of these in-
flammatory signaling pathways in LPS-injected mouse brains (Fig. 8).
Our results indicated that LPS treatment increased the expression of
TLR4, p-P65, p-P38, p-JNK, and p-ERK (Fig. 8b–c, f–g), but had no
evident effect on the expression of p-AKT (Fig. 8e). Castration sig-
nificantly promoted the LPS-induced activation of these proteins in the
brain but these effects were abrogated by DHT supplementation
(Fig. 8). These results suggest that DHT modulates the TLR4-mediated
NF-κB and MAPK inflammation signaling pathways in LPS-injected
mouse brains.
3.7. Effects of DHT on behavioral activity in LPS-treated mice
Acute LPS treatment also affects movement in mice, including motor
activity and locomotor activity (Oliveira-Lima et al., 2019). Thus, we
evaluated whether DHT affected these behavioral activities in LPS-in-
jected mice. Exposure to LPS remarkably decreased the numbers of
crossing (Fig. 9a), grooming (Fig. 9b), rearing (Fig. 9c) and the total
distance moved (Fig. 9d). Moreover, castration further significantly
reduced the numbers of crossing and grooming and total distance
moved; and DHT markedly improved the total distance in LPS-treated
castrated mice (Fig. 9a–d), suggesting that DHT regulates the locomotor
activity in LPS-injected mice. In the pole test, castration and DHT
treatment had no evident impact on the time it took LPS-treated mice to
turn completely downward (Fig. 9e), but castration significantly pro-
longed the time it took the LPS-treated mice to place their four paws on
the floor which was evidently shortened by DHT administration
(Fig. 9e), indicating that DHT improves the motor coordination in mice
following LPS injection. Overall, these results indicate that DHT could
improves behavioral activities in LPS-treated mice.
3.8. Effects of DHT on Aβ deposition and neuronal damage in LPS-treated
mouse brains
Low dose LPS injection causes chronic inflammatory damage to
neurons, amyloid-β (Aβ) production, synaptic loss and neuronal apop-
tosis (Chen et al., 2019; Wu et al., 2019). We thus examined the effect
of DHT on the expression of Aβ, apoptotic and synaptic proteins in the
mouse brain. Castration accelerated the LPS-induced increase in Aβ
formation (Fig. 10a) in the hippocampus, pro-apoptotic protein cas-
pase-3 in both the hippocampus and cortex compared to those of con-
trol group, as well as enhanced the decrease in the ratio of anti- to pro-
apoptotic molecules (Bcl-2/Bax) in the cortex (Fig. 10b), but DHT
treatment significantly blocked the castration-mediated amplification
effect in LPS-injected mouse brains (Fig. 10a–b). In addition, LPS

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L. Yang, et al. Neurobiology of Disease 140 (2020) 104814

Fig. 9. Effects of DHT on locomotor activity and motor coordination in LPS-treated mice. a) Crossing, b) grooming, c) rearing activity and d) total distance
moved of the mice in the open field task. e) The time it took each mouse to turn completely downward and f) to place its four paws on the floor were determined in
the pole test. The data are expressed as the mean ± SD (n = 8). #P < .05, ###P < .001, the control group vs. the LPS group; $$P < .01, $$$ P < .001, the
castration + LPS group vs. the castration + DHT + LPS group; **P < .01, *** P < .001 vs. the control group; NS: no significance; One-way ANOVA analysis of
variance.

markedly reduced the protein level of synaptophysin (Fig. 10c) in the
brain, which was further decreased by castration in the cortex but had
no effect in the hippocampus. DHT supplementation markedly reversed
the effect of castration both in the hippocampus and cortex (Fig. 10c).
Consistently, Nissl staining revealed that LPS treatment caused neu-
ronal loss in the hippocampus and cortex (Fig. 10d). In castrated mice,
LPS further reduced the numbers of neurons in the cortex, but DHT
administration significantly increased neuronal survival in both hip-
pocampus and cortex (Fig. 10d). These results suggest that DHT reg-
ulates Aβ deposition and protects against LPS-induced synaptic damage
and neuronal loss in the mouse brain.
3.9. Effects of DHT on cognitive function in LPS-treated mice
after castration significantly decreased the time taken by LPS-treated
castrated mice to arrive at the platform (Fig. 11a–b). When the platform
was removed on day 33, the average escape latency (Fig. 11c), time
spent in the target quadrant (Fig. 11d) and crossing numbers over the
platform site (Fig. 11e) in LPS-treated mice were significantly pro-
longed compared with those of the control group. However, castration
had no evident effect on these parameters, whereas DHT administration
markedly improved all of these parameters in LPS-treated castrated
mice (Fig. 11c–e). These behavioral parameters were not affected by
motor function, because there was no significant difference in the
average speed of these mice among the five groups (Fig. 11d). Taken
together, these results suggest that DHT ameliorates the LPS-induced
spatial and learning impairment.

LPS-induced neuronal damage contributes to cognitive impairment 4. Discussion

(Zhao et al., 2019). We investigated the effect of DHT on spatial and
learning behavior in LPS-treated mice by using the MWM test. The time
course of the learning curve revealed that LPS-injected mice arrived at
the hidden platform significantly more slowly than the control mice
from day 29 to day 32, and castration prolonged the time to reach the
location of the platform in LPS-injected mice, although there was not
significant difference (Fig. 11a–b). However, DHT supplementation
This is the first study to report that DHT exerts anti-neuroin-
flammatory effects in vitro and in vivo by inhibiting microglial acti-
vation and the release of proinflammatory factors, attenuating neuronal
damage, and ultimately ameliorating cognitive impairment and motor
dysfunction in LPS-induced neuroinflammation.
Aberrant glial activation and neuroinflammation are believed to

10
L. Yang, et al. Neurobiology of Disease 140 (2020) 104814

Fig. 10. Effects of DHT on amyloid-β formation and neuronal damage in LPS-treated mouse brains. Wild-type C57BL/6 mice were injected with DHT (5 mg/kg,
s.c.) or oil daily for 15 days, followed by injection with LPS (1 mg/kg, i.p.) or saline for 5 days. a) Aβ, b) Bcl-2, Bax, Caspase 3, and c) synaptophysin protein levels
were detected by Western blotting (n = 4). d) Neuronal morphology was detected by Nissl staining (n = 5). *P < .05, **P < .01 and *** P < .001 vs. the control
group; #P < .05, ##P < .01 and ###P < .001 vs. the LPS group; $P < .05, $$P < .01 and $$$P < .001 vs. the Castration + LPS group; NS: no significance; One-
way ANOVA analysis of variance.

play a prominent role in the pathogenesis of neurodegenerative dis-
eases. Previous studies indicated that androgens suppress macrophage
activation and inflammatory responses (Corcoran et al., 2010;
D'Agostino et al., 1999). Therefore, DHT may possess anti-inflammatory
properties in the brain. Activated microglia release inflammatory fac-
tors such as NO, PGE2, TNF-α and IL-1β upon LPS stimulation (Zhou
et al., 2020). Androgen replacement therapy attenuates the peripheral
inflammatory process by inhibiting the expression and function of in-
flammatory cytokines including TNF-α, IL-1β, and IL-6 (Corrales et al.,
2009; Vignozzi et al., 2012). Here, we found that DHT suppressed the
production of inflammatory factors including iNOS, COX-2, NO, PGE2,
TNF-α, IL-1β and IL-6 in LPS-stimulated microglia, and these
inflammatory mediators were modulated by DHT in LPS-treated mouse
brains. DHT also regulated the mRNA expression of the anti-in-
flammatory factors IL-10 and IL-13. These findings were in consistent
with previous studies showing that androgen treatment inhibited the
expression of NO, TNF-α and IL-1β, as well as increased the synthesis of
IL-10 under inflammatory conditions (D'Agostino et al., 1999; Malkin
et al., 2004). Thus, DHT exerts the anti-inflammatory function by in-
hibiting microglial activation. Moreover, castration alone failed to ac-
tivate microglia or elicit the release of inflammatory factors, suggesting
androgen deficiency alone does not induce an inflammatory response in
brain but exacerbate preexisting inflammatory conditions.
How does DHT downregulate proinflammatory mediators? Previous

11
L. Yang, et al. Neurobiology of Disease 140 (2020) 104814

Fig. 11. Effects of DHT on LPS-induced cognitive impairment in wild-type C57BL/6 mice. a) A time course learning curve showing the latency to locate the
platform from day 28 to day 32. b) The hidden platform was marked in green in the water maze. Representative searching swimming tracks of the mice in the probe
trial test. c) Escape latency to reach the location of the removed platform on day 33. d) Time spent in the platform quadrant, e) number of crossings over the location
of the removed platform during, f) average speed in the probe test. Data are expressed as the mean ± SD (n = 8). #P < .05, ##P < .01, the control group vs. the
LPS group; $$P < .01, $$$ P < .001, the castration + LPS group vs. the castration + DHT + LPS group; *P < .05, **P < .01, ***P < .001 vs. the control group;
NS: no significance; One-way ANOVA analysis of variance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

studies have demonstrated that androgen treatment of macrophages
and endothelial cells in vitro decreased the expression of TLR4 at the
transcriptional and translational levels (Norata et al., 2006, 2010;
Rettew et al., 2008), and androgen supplementation abolished the in-
crease in TLR4 protein expression of castrated animals (Leimgruber
et al., 2013). Thus, DHT might suppress the expression of TLR4 on the
cell surface and deactivate downstream signaling pathways to inhibit
the neuroinflammatory response. LPS binds to TLR4 and activates
downstream inflammatory signaling cascades including NF-κB and
MAPKs (Lu et al., 2008). Consistent with previous findings that DHT
inhibits the expression of NF-κB and the subsequent inflammatory re-
sponse (Gonzales et al., 2009; Norata et al., 2006), we further observed
that, in LPS-stimulated microglia, DHT inhibited the activation of the
NF-κB p65 and MAPK p38 pathways, which was primarily TLR4-de-
pendent. Furthermore, DHT regulated the phosphorylation of p65, p38,
ERK and JNK, but had no effect on the expression of AKT in LPS-treated
mouse brains. In parallel, several studies have reported that DHT sup-
presses ERK and p38 signaling in vitro and in vivo (Chen et al., 2016; Su
et al., 2015; Yao et al., 2017). Take together, DHT inhibits the neu-
roinflammatory response though the TLR4-mediated NF-κB and MAPK
signaling pathways.
Chronic neuroinflammation mediates neuronal damage and apop-
tosis in the pathogenesis of neurodegenerative diseases, including AD
and PD. As an important component of senile plaques in AD, Aβ is a
causative factor and induces neuronal loss through activation of
apoptotic pathways (Lei and Renyuan, 2018). A study reported that the
upregulation of Aβ1–42 was accompanied by an increase in caspase-3
and Bax, and a decrease in Bcl-2 in systemic LPS-injected mouse brains
(Chen et al., 2017; Wu et al., 2019). Analogous to these results, we
observed that LPS treatment resulted in increases in Aβ1–42, caspase-3
and the ratio of Bax/Bcl-2 in the brain, and DHT modulated the ex-
pression of these proteins, suggesting anti-amyloidogenic and anti-
apoptotic effects of DHT in LPS-induced neuroinflammation. The me-
chanism by which DHT reduces LPS-mediated Aβ deposition and neu-
ronal apoptosis might be that by regulating the activity of Aβ hydro-
lases and degradation enzymes, as well as by facilitating microglial
phagocytosis of Aβ (Lei and Renyuan, 2018). In addition, synapto-
toxicity of LPS was reported to be associated with the synaptic protein
synaptophysin, which is implicated in synaptic plasticity and cognitive
function (Rehman et al., 2019; Zhou et al., 2020). In the present study,
we showed that LPS administration decreased synaptophysin in the
brain, which was regulated by DHT, indicating that DHT mitigates LPS-
induced synaptic damage. Further Nissl staining revealed that DHT also
attenuated the decrease in neurons in the hippocampus and cortex of
LPS-induced mouse brains. Therefore, these results suggest that DHT
exerts neuroprotective effects in LPS-induced neuroinflammation.
Neuronal loss in the hippocampus and cortex is closely related to
cognitive and behavioral dysfunction. Accumulating evidence indicates
that systemic LPS injection in mice induces cognitive deficits, including
spatial learning and memory impairment, as measured by MWM, which

12
L. Yang, et al.
is the most popular test of hippocampal-dependent cognitive functions
(Chen et al., 2019; Zhao et al., 2019). Moreover, age-related depletion
of androgens is associated with cognitive decline in elderly men and
androgen supplementation is beneficial for cognitive function
(Drummond et al., 2009; Mohamad et al., 2018). In the current study,
using the MWM, we observed that LPS treatment led to increased es-
cape latency, a reduced proportion of time spent in the target quadrant
and reduced platform crossing times. DHT administration improved
these parameters in LPS-treated castrated mice, suggesting that DHT
improves cognitive impairments in LPS-treated castrated mice. Our
results also supported the observation that castration alone has no ef-
fect on the visible platform test and swimming speed in the MWM
(Hajali et al., 2015). In addition, previous studies have shown that LPS
administration in mice affects locomotor and motor activity (Kim et al.,
2019a,b). Herein, our results showed that acute LPS injection reduced
the locomotor activity and motor willingness in castrated mice, which
were improved by DHT. Pole test also suggested that DHT supple-
mentation in castrated mice had benefits on LPS-induced motor in-
coordination. One explanation might be that DHT protects acet-
ylcholinesterase neurons from LPS challenge, but the mechanism by
which DHT improves LPS-induced motor dysfunction is unclear and
requires further investigation. Take together, these results indicate that
DHT improves the cognitive deficits and behavioral disturbance.
5. Conclusions
We demonstrated that DHT ameliorates neuronal apoptosis and
improves cognitive and behavioral impairments in LPS-induced neu-
roinflammation. The mechanisms of the neuroprotective effects of DHT
are through inhibiting of inflammatory responses induced by LPS-ac-
tivated microglia via the TLR4-mediated NF-κB and MAPK signaling
pathways. Therefore, this study suggests that androgen replacement
therapy is a potential therapeutic strategy for improving cognitive
function in neuroinflammation-related diseases.
Grant numbers and resources
None.
Declaration of Competing Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by the Jing'an District Central Hospital
Research Grant, Shanghai, China. We thank Professor Huang Fang
(Brain Institute, Fudan University, Shanghai, China) and her research
group for their assistance in experimental technologies and materials.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.nbd.2020.104814.
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