Behavioural Brain Research 322 (2017) 1–8

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Behavioural Brain Research

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Research report

NLRP3 gene knockout blocks NF-␬B and MAPK signaling pathway in

CUMS-induced depression mouse model
Wen-Jun Su a,1 , Yi Zhang a,b,1 , Ying Chen c , Hong Gong a , Yong-Jie Lian a , Wei Peng a ,
Yun-Zi Liu a , Yun-Xia Wang a , Zi-Li You d , Shi-Jie Feng e , Ying Zong e , Guo-Cai Lu e,∗ ,
Chun-Lei Jiang a,∗
a
Laboratory of Stress Medicine, Faculty of Psychology and Mental Health, Second Military Medical University, 800 Xiangyin Road, Shanghai, China
b
Department of Psychiatry, Faculty of Psychology and Mental Health, Second Military Medical University, 800 Xiangyin Road, Shanghai, China
c
Department of Pharmacy, The 81st Hospital of PLA, 34 Yanggongjing, Nanjing, China
d
School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, 610054, China
e
New Drug Safety Evaluation Center, Second Military Medical University, 800 Xiangyin Road, Shanghai, China

h i g h l i g h t s

• CUMS-induced depression-like behavior needs participation of NLRP3 inflammasome.

• NLRP3 gene knockout blocks activation of NF-␬B protein complex in CUMS-induced depression mouse model.
• The NLRP3 inflammasome regulates CUMS-induced MAPK pathway activation.

a r t i c l e i n f o a b s t r a c t

Article history: Background: Abundant researches indicate that neuroinflammation has important roles in the pathophys-
Received 1 June 2016 iology of depression. Our previous study found that the NLRP3 inflammasome mediated stress-induced
Received in revised form depression-like behavior in mice via regulating neuroinflammation. However, it still remains unclear
20 December 2016
that how the NLRP3 inflammasome influences related inflammatory signaling pathway to contribute to
Accepted 10 January 2017
neuroinflammation in depression.
Available online 16 January 2017
Methods: We used wild-type mice and NLRP3 gene knockout mice to explore the role of NLRP3 inflamma-
some and related inflammatory signaling pathways in chronic unpredictable mild stress (CUMS) induced
Keywords:
NLRP3 inflammasome
depression mouse model.
Knockout Results: Both wild-type and NLRP3 knockout stress group mice gained less weight than control group mice
Interleukin-1␤ after 4 weeks CUMS exposure. The wild-type mice subjected to 4 weeks CUMS displayed depression-like
Depression behaviors, including decreased sucrose preference and increased immobility time in the tail suspension
Stress test. The NLRP3 knockout stress group mice didn’t demonstrate depression-like behaviors. The levels of
Inflammation interleukin-1␤ protein in serum and hippocampi of CUMS exposed wild-type mice were significantly
higher, while the NLRP3 knockout stress group mice didn’t show an elevation of interleukin-1␤ levels.
Similarly to early researches, we found that CUMS led to promoted NLRP3 expression in hippocampi. In
addition, the hippocampi in CUMS exposed wild-type mice had higher p-JNK and p-p38 protein expres-
sion, which indicated activation of the mitogen-activated protein kinases (MAPK) pathway. The knockout
of NLRP3 gene inhibited CUMS-induced activation of the MAPK pathway. The nuclear factor kappa-light-
chain-enhancer of activated B cells (NF-␬B) protein complex was activated in the hippocampi of wild-type
mice after CUMS exposure, while knockout of NLRP3 gene hindered its activation.
Conclusions: These data further proved that the NLRP3 inflammasome mediated CUMS-induced
depression-like behavior. The NLRP3 inflammasome regulated CUMS-induced MAPK pathway and NF-
␬B protein complex activation in depression mouse model. Further researches targeting the NLRP3
inflammasome-signaling pathway might be under a promising future in the prevention and treatment
of depression.
© 2017 Elsevier B.V. All rights reserved.

∗ Corresponding authors.
E-mail addresses: newdrug@smmu.edu.cn (G.-C. Lu), cljiang@vip.163.com (C.-L. Jiang).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.bbr.2017.01.018
0166-4328/© 2017 Elsevier B.V. All rights reserved.
2 W.-J. Su et al. / Behavioural Brain Research 322 (2017) 1–8
1. Introduction
Major depressive disorder (MDD) characterizes by depressed
mood most of the day or markedly diminished interest or pleasure
in all activities most of the day [1]. As a pervasive mental disor-
der and major contributor to the global burden of diseases, there
are about 350 million people suffering from MDD across the world
[2,3]. The prevalence of MDD for lifetime was 16.2% in US (32.6-35.1
million US adults), and the overall estimation of lifetime prevalence
of MDD was 3.3% in mainland of China [4,5]. As it has huge social
and economic importance, researchers around the world are try-
ing to figure out the fundamental pathophysiological mechanisms
of MDD. After decades of extensive research, researchers postulate
several hypotheses potentially underlying the MDD progression,
such as the neurotransmitter hypothesis, neuronal circuit hypoth-
esis and proinflammatory cytokine hypothesis.
The proinflammatory cytokine hypothesis suggests that inflam-
matory cytokines including tumor necrosis factor-␣ (TNF-␣),
interleukin-6 and interleukin-1␤ (IL-1␤), contribute to neuroin-
flammation and suppress neurogenesis in the central nervous
system [6–9]. Reichenberg A et al. reported that in human, endo-
toxin administration resulted in transient significant increase in
the levels of anxiety and depressed mood [10], while Goshen et al.
found that in mice, elevated levels of brain IL-1 were causally
related to various aspects of depression-like behavior, including the
behavioral symptomatology, adrenocortical activation and reduced
neurogenesis [11]. It is notable that maturation and secretion of
IL-1␤ is dependent upon the activation of NLRP3 inflammasome,
which is a multi-protein complex of the innate immune system,
and serves as an upstream regulator of the IL-1␤ signaling path-
way [12–14]. Iwata et al. postulated that the NLRP3 inflammasome
might be a novel target for the development of treatments for MDD
[15]. Our previous studies suggested that the NLRP3 inflammasome
mediated lipopolysaccharide (LPS) and chronic unpredictable mild
stress (CUMS) induced depression-like behaviors in mice [16,17].
Alcocer-Gomez et al. indicated that stress-induced depression-like
behaviors required a functional NLRP3 inflammasome [18].
There are three major models for NLRP3 inflammasome acti-
vation: (1) NLRP3 inflammasome agonist adenosine triphosphate
(ATP) triggers activation of the purinergic type 2X7 receptor
(P2X7R) and leads other extracellular agonists to entering the
cytosol; (2) Danger-associated molecular patterns (DAMPs) and
pathogen-associated molecular patterns (PAMPs) lead to NLRP3
inflammasome formation via the reactive oxygen species (ROS)
dependent pathway; (3) The engulfed crystalline or particulate
NLRP3 inflammasome agonists can be sensed in the cytoplasm
after lysosomal rupture [12]. Iwata et al. reported that the
ATP/P2X7R-NLRP3 inflammasome cascade in the brain could sense
the psychological stress, which led to anhedonic and anxious
behaviors [19]. Our previous study found that blocking genera-
tion of ROS in the hippocampus and prefrontal cortex (PFC) with
antioxidative hydrogen-rich water could inhibit inflammasome
activation and prevent the development of depression-like behav-
iors induced by CUMS [20].
As an important contributor to the pathogenesis of MDD, ROS
might exert effects on inflammatory pathways via activating the
nuclear factor kappa-light-chain-enhancer of activated B cells (NF-
␬B) and mitogen-activated protein kinases (MAPK) family stress
kinases [21]. Bioinformatics analysis found that the MAPK path-
way was one of the functionally enriched cell-signaling pathways
engaged in MDD [22]. Since NF-␬B could interact with a bunch of
genes involved in inflammation, researchers observed activation of
NF-␬B in many inflammatory diseases, such as arthritis, asthma and
atherosclerosis [23]. Song et al. found that NF-␬B was associated
with schizophrenia through regulating the expression of cytokines
in patients’ serum [24]. Several studies indicated that the NF-␬B sig-
naling cascade might have particular relevance to MDD [21,25,26].
In an integrative rat-human study identifying genes and gene path-
ways associated with MDD, researchers found that eighty percent
of those uncovered genes that might have a role in the pathogenesis
of MDD were functionally associated with stress response signaling
cascade, involving NF-␬B and MAPK pathway [27].
However, it is not clear if and how the NLRP3 inflamma-
some influences the NF-␬B and MAPK pathway to contribute
to neuroinflammation in MDD. We hypothesize that the NLRP3
inflammasome pathway may exert its effects on MDD via reg-
ulating the NF-␬B and MAPK stress response signaling cascade.
In this study, both wild-type and NLRP3 gene knockout mice are
subjected to CUMS treatment. After 4-week stress exposure, we
perform depression-like behavior tests and measure IL-1␤ levels,
NLRP3 expression, NF-␬B activation and MAPK pathway protein
expression to explore if and how NLRP3 gene knockout influence
the NF-␬B and MAPK inflammatory pathway in MDD animal model.
As we expected, the results suggested that the NLRP3 inflamma-
some contributed to neuroinflammation through regulating NF-␬B
and MAPK inflammatory signaling pathways in MDD.
2. Materials and methods
2.1. Experimental animals
Male wild-type 8-week-old and age-matched male NLRP3 gene
knockout C57BL/6 mice (NLRP3 KO) were all introduced from New
Drug Safety Evaluation Center, Second Military Medical Univer-
sity (Shanghai, China) [28,29]. Mice were housed in a standardized
animal room (lights on 7a.m.–7p.m.; room temperature 22 ± 2 ◦ C),
with food and water provided ad libitum. Before stress procedures,
mice were acclimated to the environment and 1% sucrose solu-
tion (weight/volume) for 14 days and then randomly assigned to 4
groups, including wild-type control group, wild-type CUMS group,
NLRP3 KO control group and NLRP3 KO CUMS group (n = 10–11).
The Animal Care and Use Committee of the Second Military Medical
University approved the experiment protocols.
2.2. Chronic unpredictable mild stress protocol
The chronic unpredictable mild stress protocol (CUMS) used in
the experiment was adapted from our former published papers
[16,20]. In brief, it was a randomized 4-week schedule which con-
tained various of stressors: immobilization for 2 h; 45◦ cage tilting
for 14 h; wet bedding for 16 h; continuous illumination for 24 h;
water or food deprivation for 24 h; cage shaking for 10 min; swim-
ming in 4 ◦ C water for 5 min; 45 ◦ C heat stress for 5 min. Both the
wild-type CUMS group mice and NLRP3 KO CUMS group mice were
exposed to the aforementioned CUMS treatment, with the same
stressor applied simultaneously on the same day. In addition, no
single stressor was conducted for two consecutive days. Weight of
the mice were measured weekly at a fixed time.
2.3. Behavioral tests
Sucrose preference test is a common method to assess animal
depression-like behavior, especially anhedonia, the core symptom
of depression. In this experiment, sucrose preference test was car-
ried out at the dark phase according to our previous published
papers [16,20]: after food and water deprivation for 24 h, each ani-
mal would be provided with two bottles at the same time, one
containing tap water and the other containing 1% sucrose solution.
Fluid consumption was monitored for 1 h, and then the bottles were
removed and weighed. The sucrose preference ratio was calculated
as the percentage of sucrose solution consumption out of the sum
of tap water and sucrose solution consumption.
 W.-J. Su et al. / Behavioural Brain Research 322 (2017) 1–8 3

Fig. 1. Polyacrylamide gel electrophoresis-based genotyping analysis. Polyacrylamide gel electrophoresis method was used to genotype NLRP3 gene knockout mice acquired
from the New Drug Safety Evaluation Center. Based on NLRP3 PCR primers and mutant positions, different DNA pattern occurred after T7E1 digestion: wild-type band of
366 bp; mutant band of approximately 226 + 140 bp. NLRP3 F2 3–9 was homozygous and only one band occurred after self-hybridization, but showed two bands after DNA
hybridization with the wild-type one; while NLRP3 F1 was heterozygous, two bands appeared after both self-hybridization and hybridization with wild type mice.

Tail suspension test is another widely used behavioral param-
eter to evaluate depression-like behavior, indicating the state of
behavioral despair or helplessness. Similarly, this test was also
performed during the dark phase at the end of the whole CUMS
procedures. Each mouse was hung upside down on the hook of the
PHM-300 tail suspension chamber (MED Associates Inc., St. Albans,
VT), a standard three-walled rectangular compartment, with its
width and depth sufficiently sized to ensure that the mouse cannot
reach the walls. The suspension hook used to suspend the tail of
each mouse was positioned on the top of the chamber and linked
to mechanical sensors. After 1 min adaptation to the apparatus, the
immobility time during the next 5 mins’ suspension was recorded
and analyzed using the software, the lower threshold value was set
at 0.05. 75% ethanol was used to clean the feces and wipe the appa-
ratus between each trial to effectively eliminate influences of fecal
or urinary odor on the behavioral measurements.
2.4. Sacrifice and sample preparation
At the end of 4 weeks long CUMS procedure, 12 h after the behav-
ioral tests were completed, approximately 24 h after the last acute
stress manipulation, all the animals were sacrificed under general
anesthesia with pentobarbital. The blood was collected, coagulated
for 30 min at room temperature and subsequently centrifuged at
4000 rpm, 4 ◦ C for 15 min to get supernatant serum. Whole brain
and hippocampus were collected or dissected immediately after
decapitation, flash frozen in liquid nitrogen, and stored at − 80 ◦ C
for further analysis. It is worth mentioning that 3 whole brain sam-
ples of each group would be applied in frozen section to perform
immunofluorescence analyses, while the rest brains were only used
for Enzyme-Linked Immunosorbent Assay and Western Blotting, so
we just preserve the hippocampi.
2.5. Interleukin-1ˇ detection
IL-1␤ levels in serum and hippocampus homogenates were
measured using mouse IL-1 beta/IL-1F2 Quantikine ELISA kit
according to the manufacturer’s instructions (R&D Systems, Min-
neapolis, MN, USA). The hippocampi were homogenized in RIPA
buffer containing 1 mM PMSF protease inhibitor (Beyotime Insti-
tute of Biotechnology, Nantong, Jiangsu, China) and PhosSTOP
(Roche, Indianapolis, IN, USA) according to standard protocol. The
supernatants were collected after the hippocampus lysates were
centrifuged at 10,000g for 15 min at 4 ◦ C. The total protein concen-
trations of supernatants were determined with the Enhanced BCA
Protein Assay Kit (Beyotime Institute of Biotechnology, Nantong,
Jiangsu, China). The final results of hippocampus IL-1␤ levels were
normalized to the total protein concentration of each hippocampus
supernatant accordingly.
2.6. Western blotting
After the aforementioned ELISA detection, the rest hippocampal
homogenized lysates were added with 5X loading buffer (Bey-
otime Institute of Biotechnology, Nantong, Jiangsu, China) at 4:1
vol ratio, then mixed and heated to 100 ◦ C for 10 min followed
by cooling to 4 ◦ C and preserved at −80 ◦ C. Before each blot-
ting process, the prepared denatured specimen would be heated
again and remixed thoroughly using vortex. Western blot pro-
tocol was adapted from our previous research [16]. In brief,
denatured hippocampal supernatant samples were separated by
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(Beyotime Institute of Biotechnology, Nantong, Jiangsu, China),
transferred onto Immobilon-P Polyvinylidene Fluoride membranes
(Millipore, Bedford, MA, USA), using a wet transfer system (Bio-
Rad, Hercules, CA, USA). After blocking with 3% bovine serum
albumin (BSA) at room temperature for 1 h, the membranes were
incubated with specific primary antibodies (dilution: 1:1000) at
4 ◦ C overnight. After washing for 3 times, the membranes were
incubated with IRDye conjugated secondary antibodies (dilution:
1:5000) at room temperature for 1 h. Then the membranes were
scanned, and the densitometry values of bands was quantified with
Odyssey Infrared Imaging System (LI-COR, Inc., Lincoln, NE, USA)
and Image J Software (National Institute of Mental Health, USA).
Stripping and reprobing western blot procedure were carried out
to detecte MAPKs and NF-␬B p-65 subunit by using NewBlot IR
Stripping Buffer (LI-COR, Inc., Lincoln, NE, USA) following the man-
ufacturer’s instruction.
Primary antibodies Phospho-MAPK Family Antibody Sampler
Kit (#9910) which contained phospho-p44/42 MAPK (p-ERK 1/2),
4 W.-J. Su et al. / Behavioural Brain Research 322 (2017) 1–8

Fig. 2. CUMS induced weight, sucrose preference and tail suspension changes. Both wild-type and NLRP3 KO mice were subjected to CUMS procedure for 4 weeks. (A)
Changes in body weight. The weight gaining pace of the wild-type stress group mice tended to slow down from the second week of CUMS. Meanwhile, weight of mice from
NLRP3 KO stress group dropped suddenly in the fourth CUMS week. (B) Percentage rates of sucrose preference test after the CUMS procedure. The rates of wild-type CUMS
group mice were significantly lower than that of the non-stressed wild-type group. The knockout mice didn’t demonstrate such difference. (C) Immobility time during the
tail suspension test after the CUMS procedure. Similarly, immobility durations of mice in wild-type CUMS group were obviously longer than those from non-stressed group.
NLRP3 knockout eliminated the phenotype. All of the changes indicate depression-like behavior of the wild-type CUMS group mice, but not mice in the NLRP3 KO CUMS
group. Data were shown as mean ± SEM (n = 10 for each group), *p < 0.05 compared to wild-type control group mice.

phospho-p38 MAPK(p-p38), phospho-SAPK/JNK(p-JNK) antibod-
ies and MAPK Family Antibody Sampler Kit (#9926) containing
ERK1/2, p38 and JNK antibodies were purchased from Cell Signaling
Technology, Inc. (CST, Beverly, MA, USA). Goat anti-mouse NLRP3
polyclonal antibody (ab4207) were bought from Abcam plc. (Cam-
bridge, UK). Rabbit anti-mouse GAPDH antibody was purchased
from Sangon Biotech Co., Ltd. (Shanghai, China). Rabbit anti-mouse
NF-␬B subunit p65 (AB41733-1) and phospho-p65(Ser536) (p-p65)
antibodies (AB11014-1) were both bought from Abscitech (Balti-
more, MD, USA). IRDye 800CW donkey anti-goat and IRDye 800CW
goat anti-rabbit secondary antibodies were both purchased from
LI-COR Biosciences (LI-COR, Inc., Lincoln, NE, USA).
2.7. Statistical analysis
The data were presented as mean ± standard error (SEM) and
differences considered statistically significant only when p-values
are less than 0.05. Data was mainly analyzed using a two-way
analysis of variance (ANOVA) followed by the Bonferroni’s multi-
ple comparisons post hoc test with GraphPad Prism 6 (GraphPad
Software, Inc., La Jolla, CA, USA). Two-way ANOVA for repeated
measures was used to analyze the weight curves. Student’s t-test
was applied to detect significant difference between two groups,
namely NLRP3 protein level analysis.
3. Results
3.1. Polyacrylamide gel electrophoresis-based genotyping
analysis
In order to genotype NLRP3 gene knockout mice, a poly-
acrylamide gel electrophoresis-based method was applied. F2
generation homozygous NLRP3 knockout mice were adopted dur-
ing the mating process of F1 generation heterozygote mice and
genotyped by T7E1 digestion method. Based on NLRP3 PCR primers
and mutant positions, different DNA pattern occurred after T7E1
digestion: wild-type band of 366 bp; mutant band of approximately
226 + 140 bp. NLRP3 F2 3–9 was homozygous and occurred only
one band after self-hybridization, but showed two bands after DNA
hybridization with the wild-type one; while NLRP3 F1 was het-
erozygous, two bands appeared after both self-hybridization and
hybridization with wild-type mice (Fig. 1). Considering the gene
backgrounds, homozygous descendent NLRP3 knockout mice were
taken into account to carry out the entire research plan, while wild-
type mice are non-littermate.
3.2. NLRP3 gene knockout mice did not demonstrate
depression-like behaviors after 4-weeks CUMS
In order to evaluate the effect of CUMS on each group, the
weights of mice were measured weekly, behavioral test including
the sucrose preference test and the tail suspension test were carried
out at the end of the CUMS protocol. Generally, during the CUMS
procedure, mice in all the four groups (n = 10) gained weight week
after week. As is presented, after two weeks’ CUMS manipulation,
the weight gaining pace of the wild-type stress group mice tended
to slow down, while mice in the wild-type control group main-
tained their increasing trend to the end of the CUMS treatment.
Interestingly, both stress and unstressed group NLRP3 knockout
mice put on weight in a generally similar manner in the first three
weeks, whereas in the last week, the weight of the NLRP3 knockout
stress group mice dropped in a sudden (Fig. 2A). In addition to the
weight change, the wild-type mice subjected to 4 weeks’ CUMS also
displayed depression-like behaviors, including decreased sucrose
preference (Fig. 2B, n = 10, *p < 0.05, F(1, 36) = 0.5476, t = 2.735) and
increased immobility time in the tail suspension test when com-
pared to wild-type control group mice (Fig. 2C, n = 10, *p < 0.05, F(1,
36) = 3.243, t = 2.343). However, the NLRP3 knockout stress group
mice didn’t demonstrate such depression-like behaviors (Fig. 2B
and C).
 W.-J. Su et al. / Behavioural Brain Research 322 (2017) 1–8 5

Fig. 3. Measurement of IL-1␤ levels. (A) Serum IL-1␤ levels were significantly higher in CUMS exposed wild-type mice than that of the wild-type control group mice, while
no such elevation was observed in the NLRP3 knockout group mice. (B) Protein levels of IL-1␤ in hippocampi were normalized to the total protein levels of each hippocampal
supernatant sample respectively. Hippocampal IL-1␤ protein level of wild-type mice subjected to CUMS was significantly higher comparing with wild-type control group
mice, no such increased IL-1␤ protein level was produced in that of NLRP3 knockout mice. All the results were shown as mean ± SEM (n = 8-9 for A, n = 5 for B), **p < 0.01
compared to wild-type control group mice.

Fig. 4. CUMS induced NLRP3 expression in hippocampi. (A) Western blots were used to detect the expression of NLRP3 protein. (B) Densitometric measurements which
stand for protein levels of NLRP3 were normalized to that of GAPDH. Statistical analyses indicated that CUMS could lead to significantly promoted expression in hippocampi
of wild-type mice. Results were demonstrated as mean ± SEM (n = 3 for each group), *p < 0.05 when compared to wild-type control group mice.

3.3. NLRP3 gene knockout abrogated elevation of both serumal 3.5. CUMS prompted phosphorylation of NF-B subunit p65,
and hippocampal IL-1ˇ levels induced by CUMS while NLRP3 gene knockout could block it

To determine the inflammatory situation especially which
induced by NLRP3 inflammasome activation, the classical proin-
flammatory cytokine IL-1␤ in both peripheral and central nervous
system were detected respectively. As is displayed, the serumal
IL-1␤ protein levels were significantly higher in CUMS exposed
wild-type mice than that of the wild-type control group mice, while
the NLRP3 knockout stress group mice didn’t show an elevation of
IL-1␤ levels (Fig. 3A, n = 8–9, **p < 0.01, F(1, 29) = 23.32, t = 3.632).
At the meantime, the hippocampi of CUMS treated wild-type mice
contained significantly higher levels of IL-1␤ protein when com-
pared to wild-type control group mice, however, no such increase
in hippocampal IL-1␤ protein level was detected in NLRP3 knockout
mice subjected to CUMS (Fig. 3B, n = 5, **p < 0.01, F(1, 16) = 6.562,
t = 4.108). Protein levels of IL-1␤ in hippocampi were normalized
to the total protein levels of each hippocampal supernatant respec-
tively.
Considering that NF-␬B plays a pivotal role in classical inflam-
matory pathway and has complicated influence on not only
neurogenesis but also IL-1␤ priming, we checked the phos-
phorylation of NF-␬B subunit p65 to represent its activation.
Representative immunoblot bands and related statistical analyses
were displayed in Fig. 5A&B. It was suggested that phosphoryla-
tion of p65 was significantly enhanced in wild-type CUMS mice
than that of wild-type control mice (n = 3, **p < 0.01, F(1, 8) = 12.30,
t = 5.504). Strikingly, NLRP3 gene knockout mice seemed to main-
tain p65 phosphorylation at a relatively normal level, even after
4-weeks CUMS. The above results indicated that knockout of NLRP3
inflammasome hindered NF-␬B protein activation resulted from
CUMS exposure.
3.6. CUMS-induced MAPK signaling pathway expression in
hippocampi

3.4. CUMS led to promoted NLRP3 expression in hippocampi
According to our previous studies, 4 weeks’ CUMS treatment
would result in an obvious promotion of NLRP3 protein expres-
sion. That may contribute to the following assembly and activation
of NLRP3 inflammasome. Here we detected the relative protein
levels of NLRP3 from each group using western blot. Apparently,
bands of wild-type CUMS group mice got stronger signal than that
of the control group. Meanwhile, there were no visible bands in
either stressed or non-stressed NLRP3 KO group (Fig. 4A). Densit-
ometric measurements and analyses suggested that the difference
between wild-type control group and wild-type CUMS group was
statistically significant (Fig. 4B, n = 3, *p < 0.05, t = 4.435).
Although MAPK signaling pathway has various functions upon
growth and development; it is susceptible to surrounding envi-
ronment including stress and inflammation, and also affects
inflammatory balance in turn. Taking these circumstances into
consideration, western blots were applied to detect the protein
expression of MAPK family. Protein levels of p-JNK, p-ERK1/2,
and p-p38 were divided by the protein levels of JNK, ERK1/2 and
p38 respectively, before that they were normalized to the GAPDH
protein levels. All the other three groups’ protein levels were pre-
sented as fold changes to wild-type control group protein levels.
The hippocampi in CUMS exposed wild-type mice had more JNK
and p38 phosphorylation when compared to the wild-type control
group mice, which indicated activation of the MAPK pathway (n = 3,
6 W.-J. Su et al. / Behavioural Brain Research 322 (2017) 1–8

Fig. 5. CUMS induced phosphorylation of NF-␬B subunit p65 in hippocampi. (A) Western blots were used to detect the expression of p65 and phospho-p65 (Ser536) protein.
(B) Protein levels of p-p65 were divided by total p65 levels, subsequent densitometric measurements and statistical analyses suggested that CUMS significantly promoted
phosphorylation of p65 in hippocampi of wild-type mice, while NLRP3 gene knockout abort this change. Results were demonstrated as mean ± SEM (n = 3 for each group),
**p < 0.01 when compared with wild-type control group mice.

Fig. 6. CUMS induced MAPK signaling pathway expression in hippocampi. (A) Western blots were used to detect the protein expression of MAPK signaling pathway. (B)
Protein levels of p-JNK, p-ERK1/2, and p-p38 were divided by total JNK, ERK1/2 and p38 levels respectively after they were normalized to the GAPDH protein levels. The
hippocampi in CUMS exposed wild-type mice had higher p-JNK and p-p38 protein expression when compared to the wild-type control group mice. However, no such increase
was detected in gene knockout mice. All the other three groups’ protein levels were presented as fold changes to wild-type control group protein levels. The results were
shown as mean ± SEM (n = 3 for each group), *p < 0.05, **p < 0.01 when compared to wild-type control group mice.

*p < 0.05, F(1, 8) = 3.440, t = 3.574 for p-p38/p38; n = 3, **p < 0.01,
F(1, 8) = 5.523, t = 3.839 for p-JNK/JNK). However, the knockout of
NLRP3 inflammasome impeded CUMS-induced activation of the
MAPK pathway (Fig. 6).
4. Discussion
In our previous studies, we found that the NLRP3 inflammasome
signaling pathway inhibitors Ac-YVAD-CMK and VX-765 could
impede LPS-induced acute depression-like behaviors and CUMS-
induced chronic depression-like behaviors respectively [16,17].
To further investigate the functional role of NLRP3 inflamma-
some in CUMS-induced depression-like behaviors and the relevant
inflammatory signaling pathways, we compared the behavioral and
molecular parameters of wild-type and NLRP3 gene knockout mice.
We weighed the animals weekly to measure how their body
weight changed when subjected to CUMS, since weight loss could
be one of the nine specific symptoms of MDD [1]. We found that
after two weeks of CUMS treatment, the wild-type stress group
mice gained less weight than the unstressed control group mice.
This trend continued to the end of CUMS treatment, which indi-
cated the progression of depression-like behavior (Fig. 2A). The
stress group NLRP3 knockout mice didn’t show delayed weight gain
when compared to the control group mice for the first three weeks.
At the end of the CUMS treatment, the weight of the stress group
NLRP3 knockout mice was less than the control group because two
of the mice in the stress group dropped weight sharply (Fig. 2A).
Unfortunately, the wild-type mice applied in this research were not
littermate ones, we cannot definitely explain the weight variance
between wild-type and NLPR3 KO groups owing to possible slight
gene background difference. However, it did not demonstrate any
 W.-J. Su et al. / Behavioural Brain Research 322 (2017) 1–8
behavioral difference between wild-type control versus NLRP3 KO
control mice throughout this research.
Sucrose preference test is widely used to assess the “anhedo-
nia” status, and tail suspension test is broadly used to indicate
the behavioral despair status of animals [30,31]. In this study, the
results of these two tests demonstrated that after 4 weeks CUMS
treatment the wild-type mice displayed depression-like behaviors,
while the NLRP3 knockout mice were resistant to development
of depression-like behaviors (Fig. 2B and C). These data further
demonstrated that the NLRP3 inflammasome mediated CUMS-
induced depression-like behaviors.
Iwata et al. reported that acute restraint stress activated the
NLRP3 inflammasome and rapidly increased proinflammatory
cytokine IL-1␤ levels in the hippocampus [19]. Stress responses
referred to a wide array of behavioral and physiological responses,
and the hypothalamic-pituitary-adrenal (HPA) axis played key
roles in the integration of adaptive responses to stress [32]. Pro-
duction of brain IL-1 as a vital regulator of stress responses linked
stress-induced activation of the HPA axis and secretion of gluco-
corticoids [33]. Goshen et al. postulated that the HPA axis underlay
the involvement of brain IL-1␤ in stress-induced depression-like
behavior [11]. Koo et al. suggested that IL-1␤ mediated the antineu-
rogenic and depression-like behavior induced by acute and chronic
stress [34]. We detected serumal and hippocampal IL-1␤ levels to
determine if there were NLRP3 inflammasome dependent matu-
ration and release of IL-1␤ in the peripheral and central nervous
system. As we expected, the wild-type mice had significantly higher
levels of IL-1␤ in serum and hippocampi when compared to other
three groups. Knockout of the NLRP3 gene blocked the increase of
IL-1␤ in the peripheral and central nervous system (Fig. 3). Sim-
ilarly, it was also confirmed that CUMS could result in promoted
NLRP3 expression in hippocampi, which was the major component
of NLRP3 inflammasome and contributed essentially to its activa-
tion (Fig. 4). These data indicated that the release of mature IL-1␤
was dependent on the NLRP3 inflammasome activation, which was
in accordance with a former research [18].
IL-1␤ mediated CUMS-induced depression-like behavior partly
via inhibiting hippocampal neurogenesis while some antidepres-
sant drugs could increase hippocampal neurogenesis [11,35].
Hippocampal neurogenesis was a critical biological and cellular
pathogenesis of MDD, and proliferation of hippocampal neural
progenitor cells could be suppressed by stress via the NF-␬B sig-
naling pathway [34]. Besides, NF-␬B mediated the priming process
of pro-IL-1␤ induced by priming signals such as toll-like recep-
tor agonist LPS or proinflammatory cytokine tumor necrosis factor
[12]. According to former researches, NF-␬B was involved in the
activation of NLRP3 inflammasome via activating pattern recogni-
tion receptors and regulating NLRP3 expression [36,37]. However,
how the NLRP3 inflammasome might influence the NF-␬B signal-
ing pathway remains elusive. We used immunoblot measurements
to check the activation of NF-␬B in hippocampi. After subjected to
4 weeks CUMS treatment, the wild-type mice had obvious phos-
phorylation of NF-␬B subunit p65 while the NLRP3 knockout mice
sustained normal level of that in the hippocampi (Fig. 5). Accord-
ing to classical theories, transcription factors of the nuclear factor
␬ B (NF-␬B)/Rel family contained five family members in mam-
mals: RelA (p65), c-Rel, RelB, NF-␬B1 (p105/p50), and NF-␬B2
(p100/p52). Heterodimeric p65-p50 complex seems to be the most
abundant type of serious of homo- or heterodimeric complexes,
also they were complexed with members of the NF-␬B inhibitor
(I␬B) family and held in an inactive state in the cytoplasm. Conven-
tionally, NF-␬B was defined activated after I␬B phosphorylation
by I␬B kinases (IKKs) and subsequently degraded, thus liberat-
ing and phosphorylating the active NF-␬B complex including p65
which would translocate into the nucleus [38,39]. To date, these
results suggested that the NLRP3 inflammasome could also influ-
7
ence the NF-␬B signaling pathway reversely, and NF-␬B might
mediate the downstream inflammatory effects of NLRP3 inflam-
masome in depression-like behaviors.
Extracellular signal-regulated kinase (ERK), c-Jun amino-
terminal kinase (JNK) and p38 proteins are members of the MAPK
signal pathways. The ERKs had function in the control of cell divi-
sion, the JNKs are critical regulators of transcription and the p38
MAPKs can be activated by inflammatory cytokines and environ-
mental stressors. These proteins might be directly involved in
the process of neuronal cell death [40,41]. MAPKs phosphatase-1
(MKP-1) was a powerful negative regulator of intracellular MAPK
signaling pathways, which were associated with depression-like
behaviors or depressive symptom severity [42–44]. However, if
the NLRP3 inflammasome can influence the MAPK family signaling
pathways remains unclear. We detected JNK, ERK and p38 proteins
expression with western blot to explore if they were influenced by
the NLRP3 inflammasome. We found that the phosphorylated JNK
and p38 protein increased significantly in the hippocampus of wild-
type mice after 4 weeks CUMS exposure, while the NLRP3 knockout
mice didn’t show such trends (Fig. 6). These results demonstrated
that the NLRP3 inflammasome could influence the MAPK family
stress kinases signaling pathway, and suggested that MAPK might
mediate the downstream inflammatory effects of NLRP3 inflamma-
some in depression-like behaviors.
It is very regretful that in our study, we have exerted our utmost
to determine the particular cell type of all the above changes in NF-
␬B and MAPK signaling pathway but failed repeatedly. According
to new discoveries, Audrey Gustin et al. raised their opinion that
NLRP3 inflammasome was expressed and functional in mouse brain
microglia but not in astrocytes [45]. In a former research, Kreisel
T et al. also indicated that disturbances in microglial functioning
has an etiological role in chronic stress-induced depression [46].
However, almost at the same time, Sonja Johann et al. claimed that
NLRP3 inflammasome was expressed by astrocytes from the spinal
cord in the SOD1 mouse model [47]. Apparently, the complicated
circumstances about this issue should be paid more attention and
need much effort in further researches, including our own in-depth
projects.
5. Conclusions
In summary, these results further demonstrated that the NLRP3
inflammasome was involved in the CUMS-induced depression-like
behaviors via regulating IL-1␤ protein expression in serum and
hippocampus. The NLRP3 inflammasome could influence the NF-
␬B signaling pathway and MAPK family stress kinases signaling
pathway. These two pathways might mediate the downstream
inflammatory effects of NLRP3 inflammasome. Our study suggested
that interventions targeting the inflammasome-IL-1␤ signaling
pathways, or the NF-␬B and MAPK signaling pathways might pro-
vide promising strategies to alleviate MDD in the future.
Author’s contributions
W-JS and YZ (Yi Zhang) contributed equally to this work. C-
LJ and G-CL designed the research; S-JF and YZ bred the NLRP3
gene knockout mice. W-JS and YZ (Yi Zhang) conducted the estab-
lishment of depression mouse model; YC, HG and Y-JL assisted
behavioral measurement and sample collection; YZ (Yi Zhang) and
YC detected IL-1␤ levels in serum and hippocampi; W-JS detected
MAPK pathway protein expression and NF-␬B translocation.; WP
detected the I␬B-␣ and NLRP3 protein expression. YZ (Yi Zhang)
and W-JS analyzed all the data and wrote the manuscript text with
Y-XW, Z-LY helped with the revision of this manuscript. All authors
read and approved the final version of manuscript.
8 W.-J. Su et al. / Behavioural Brain Research 322 (2017) 1–8
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This study funded by the National Natural Science Foundation of
China (81571169, 31371200 to C-LJ), the National Instrumentation
Program of China (2013YQ190467 to C-LJ), and the Military Medical
Research Project (BWS14J021 to C-LJ).
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