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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 naling cascade might have particular relevance to MDD [21,25,26].


In an integrative rat-human study identifying genes and gene path-
Major depressive disorder (MDD) characterizes by depressed ways associated with MDD, researchers found that eighty percent
mood most of the day or markedly diminished interest or pleasure of those uncovered genes that might have a role in the pathogenesis
in all activities most of the day [1]. As a pervasive mental disor- of MDD were functionally associated with stress response signaling
der and major contributor to the global burden of diseases, there cascade, involving NF-␬B and MAPK pathway [27].
are about 350 million people suffering from MDD across the world However, it is not clear if and how the NLRP3 inflamma-
[2,3]. The prevalence of MDD for lifetime was 16.2% in US (32.6-35.1 some influences the NF-␬B and MAPK pathway to contribute
million US adults), and the overall estimation of lifetime prevalence to neuroinflammation in MDD. We hypothesize that the NLRP3
of MDD was 3.3% in mainland of China [4,5]. As it has huge social inflammasome pathway may exert its effects on MDD via reg-
and economic importance, researchers around the world are try- ulating the NF-␬B and MAPK stress response signaling cascade.
ing to figure out the fundamental pathophysiological mechanisms In this study, both wild-type and NLRP3 gene knockout mice are
of MDD. After decades of extensive research, researchers postulate subjected to CUMS treatment. After 4-week stress exposure, we
several hypotheses potentially underlying the MDD progression, perform depression-like behavior tests and measure IL-1␤ levels,
such as the neurotransmitter hypothesis, neuronal circuit hypoth- NLRP3 expression, NF-␬B activation and MAPK pathway protein
esis and proinflammatory cytokine hypothesis. expression to explore if and how NLRP3 gene knockout influence
The proinflammatory cytokine hypothesis suggests that inflam- the NF-␬B and MAPK inflammatory pathway in MDD animal model.
matory cytokines including tumor necrosis factor-␣ (TNF-␣), As we expected, the results suggested that the NLRP3 inflamma-
interleukin-6 and interleukin-1␤ (IL-1␤), contribute to neuroin- some contributed to neuroinflammation through regulating NF-␬B
flammation and suppress neurogenesis in the central nervous and MAPK inflammatory signaling pathways in MDD.
system [6–9]. Reichenberg A et al. reported that in human, endo-
toxin administration resulted in transient significant increase in 2. Materials and methods
the levels of anxiety and depressed mood [10], while Goshen et al.
found that in mice, elevated levels of brain IL-1 were causally 2.1. Experimental animals
related to various aspects of depression-like behavior, including the
behavioral symptomatology, adrenocortical activation and reduced Male wild-type 8-week-old and age-matched male NLRP3 gene
neurogenesis [11]. It is notable that maturation and secretion of knockout C57BL/6 mice (NLRP3 KO) were all introduced from New
IL-1␤ is dependent upon the activation of NLRP3 inflammasome, Drug Safety Evaluation Center, Second Military Medical Univer-
which is a multi-protein complex of the innate immune system, sity (Shanghai, China) [28,29]. Mice were housed in a standardized
and serves as an upstream regulator of the IL-1␤ signaling path- animal room (lights on 7a.m.–7p.m.; room temperature 22 ± 2 ◦ C),
way [12–14]. Iwata et al. postulated that the NLRP3 inflammasome with food and water provided ad libitum. Before stress procedures,
might be a novel target for the development of treatments for MDD mice were acclimated to the environment and 1% sucrose solu-
[15]. Our previous studies suggested that the NLRP3 inflammasome tion (weight/volume) for 14 days and then randomly assigned to 4
mediated lipopolysaccharide (LPS) and chronic unpredictable mild groups, including wild-type control group, wild-type CUMS group,
stress (CUMS) induced depression-like behaviors in mice [16,17]. NLRP3 KO control group and NLRP3 KO CUMS group (n = 10–11).
Alcocer-Gomez et al. indicated that stress-induced depression-like The Animal Care and Use Committee of the Second Military Medical
behaviors required a functional NLRP3 inflammasome [18]. University approved the experiment protocols.
There are three major models for NLRP3 inflammasome acti-
vation: (1) NLRP3 inflammasome agonist adenosine triphosphate 2.2. Chronic unpredictable mild stress protocol
(ATP) triggers activation of the purinergic type 2X7 receptor
(P2X7R) and leads other extracellular agonists to entering the The chronic unpredictable mild stress protocol (CUMS) used in
cytosol; (2) Danger-associated molecular patterns (DAMPs) and the experiment was adapted from our former published papers
pathogen-associated molecular patterns (PAMPs) lead to NLRP3 [16,20]. In brief, it was a randomized 4-week schedule which con-
inflammasome formation via the reactive oxygen species (ROS) tained various of stressors: immobilization for 2 h; 45◦ cage tilting
dependent pathway; (3) The engulfed crystalline or particulate for 14 h; wet bedding for 16 h; continuous illumination for 24 h;
NLRP3 inflammasome agonists can be sensed in the cytoplasm water or food deprivation for 24 h; cage shaking for 10 min; swim-
after lysosomal rupture [12]. Iwata et al. reported that the ming in 4 ◦ C water for 5 min; 45 ◦ C heat stress for 5 min. Both the
ATP/P2X7R-NLRP3 inflammasome cascade in the brain could sense wild-type CUMS group mice and NLRP3 KO CUMS group mice were
the psychological stress, which led to anhedonic and anxious exposed to the aforementioned CUMS treatment, with the same
behaviors [19]. Our previous study found that blocking genera- stressor applied simultaneously on the same day. In addition, no
tion of ROS in the hippocampus and prefrontal cortex (PFC) with single stressor was conducted for two consecutive days. Weight of
antioxidative hydrogen-rich water could inhibit inflammasome the mice were measured weekly at a fixed time.
activation and prevent the development of depression-like behav-
iors induced by CUMS [20]. 2.3. Behavioral tests
As an important contributor to the pathogenesis of MDD, ROS
might exert effects on inflammatory pathways via activating the Sucrose preference test is a common method to assess animal
nuclear factor kappa-light-chain-enhancer of activated B cells (NF- depression-like behavior, especially anhedonia, the core symptom
␬B) and mitogen-activated protein kinases (MAPK) family stress of depression. In this experiment, sucrose preference test was car-
kinases [21]. Bioinformatics analysis found that the MAPK path- ried out at the dark phase according to our previous published
way was one of the functionally enriched cell-signaling pathways papers [16,20]: after food and water deprivation for 24 h, each ani-
engaged in MDD [22]. Since NF-␬B could interact with a bunch of mal would be provided with two bottles at the same time, one
genes involved in inflammation, researchers observed activation of containing tap water and the other containing 1% sucrose solution.
NF-␬B in many inflammatory diseases, such as arthritis, asthma and Fluid consumption was monitored for 1 h, and then the bottles were
atherosclerosis [23]. Song et al. found that NF-␬B was associated removed and weighed. The sucrose preference ratio was calculated
with schizophrenia through regulating the expression of cytokines as the percentage of sucrose solution consumption out of the sum
in patients’ serum [24]. Several studies indicated that the NF-␬B sig- 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- tute of Biotechnology, Nantong, Jiangsu, China) and PhosSTOP
eter to evaluate depression-like behavior, indicating the state of (Roche, Indianapolis, IN, USA) according to standard protocol. The
behavioral despair or helplessness. Similarly, this test was also supernatants were collected after the hippocampus lysates were
performed during the dark phase at the end of the whole CUMS centrifuged at 10,000g for 15 min at 4 ◦ C. The total protein concen-
procedures. Each mouse was hung upside down on the hook of the trations of supernatants were determined with the Enhanced BCA
PHM-300 tail suspension chamber (MED Associates Inc., St. Albans, Protein Assay Kit (Beyotime Institute of Biotechnology, Nantong,
VT), a standard three-walled rectangular compartment, with its Jiangsu, China). The final results of hippocampus IL-1␤ levels were
width and depth sufficiently sized to ensure that the mouse cannot normalized to the total protein concentration of each hippocampus
reach the walls. The suspension hook used to suspend the tail of supernatant accordingly.
each mouse was positioned on the top of the chamber and linked
to mechanical sensors. After 1 min adaptation to the apparatus, the 2.6. Western blotting
immobility time during the next 5 mins’ suspension was recorded
and analyzed using the software, the lower threshold value was set After the aforementioned ELISA detection, the rest hippocampal
at 0.05. 75% ethanol was used to clean the feces and wipe the appa- homogenized lysates were added with 5X loading buffer (Bey-
ratus between each trial to effectively eliminate influences of fecal otime Institute of Biotechnology, Nantong, Jiangsu, China) at 4:1
or urinary odor on the behavioral measurements. 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-
2.4. Sacrifice and sample preparation ting process, the prepared denatured specimen would be heated
again and remixed thoroughly using vortex. Western blot pro-
At the end of 4 weeks long CUMS procedure, 12 h after the behav- tocol was adapted from our previous research [16]. In brief,
ioral tests were completed, approximately 24 h after the last acute denatured hippocampal supernatant samples were separated by
stress manipulation, all the animals were sacrificed under general 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
anesthesia with pentobarbital. The blood was collected, coagulated (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China),
for 30 min at room temperature and subsequently centrifuged at transferred onto Immobilon-P Polyvinylidene Fluoride membranes
4000 rpm, 4 ◦ C for 15 min to get supernatant serum. Whole brain (Millipore, Bedford, MA, USA), using a wet transfer system (Bio-
and hippocampus were collected or dissected immediately after Rad, Hercules, CA, USA). After blocking with 3% bovine serum
decapitation, flash frozen in liquid nitrogen, and stored at − 80 ◦ C albumin (BSA) at room temperature for 1 h, the membranes were
for further analysis. It is worth mentioning that 3 whole brain sam- incubated with specific primary antibodies (dilution: 1:1000) at
ples of each group would be applied in frozen section to perform 4 ◦ C overnight. After washing for 3 times, the membranes were
immunofluorescence analyses, while the rest brains were only used incubated with IRDye conjugated secondary antibodies (dilution:
for Enzyme-Linked Immunosorbent Assay and Western Blotting, so 1:5000) at room temperature for 1 h. Then the membranes were
we just preserve the hippocampi. scanned, and the densitometry values of bands was quantified with
Odyssey Infrared Imaging System (LI-COR, Inc., Lincoln, NE, USA)
2.5. Interleukin-1ˇ detection and Image J Software (National Institute of Mental Health, USA).
Stripping and reprobing western blot procedure were carried out
IL-1␤ levels in serum and hippocampus homogenates were to detecte MAPKs and NF-␬B p-65 subunit by using NewBlot IR
measured using mouse IL-1 beta/IL-1F2 Quantikine ELISA kit Stripping Buffer (LI-COR, Inc., Lincoln, NE, USA) following the man-
according to the manufacturer’s instructions (R&D Systems, Min- ufacturer’s instruction.
neapolis, MN, USA). The hippocampi were homogenized in RIPA Primary antibodies Phospho-MAPK Family Antibody Sampler
buffer containing 1 mM PMSF protease inhibitor (Beyotime Insti- 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- and mutant positions, different DNA pattern occurred after T7E1
ies and MAPK Family Antibody Sampler Kit (#9926) containing digestion: wild-type band of 366 bp; mutant band of approximately
ERK1/2, p38 and JNK antibodies were purchased from Cell Signaling 226 + 140 bp. NLRP3 F2 3–9 was homozygous and occurred only
Technology, Inc. (CST, Beverly, MA, USA). Goat anti-mouse NLRP3 one band after self-hybridization, but showed two bands after DNA
polyclonal antibody (ab4207) were bought from Abcam plc. (Cam- hybridization with the wild-type one; while NLRP3 F1 was het-
bridge, UK). Rabbit anti-mouse GAPDH antibody was purchased erozygous, two bands appeared after both self-hybridization and
from Sangon Biotech Co., Ltd. (Shanghai, China). Rabbit anti-mouse hybridization with wild-type mice (Fig. 1). Considering the gene
NF-␬B subunit p65 (AB41733-1) and phospho-p65(Ser536) (p-p65) backgrounds, homozygous descendent NLRP3 knockout mice were
antibodies (AB11014-1) were both bought from Abscitech (Balti- taken into account to carry out the entire research plan, while wild-
more, MD, USA). IRDye 800CW donkey anti-goat and IRDye 800CW type mice are non-littermate.
goat anti-rabbit secondary antibodies were both purchased from
LI-COR Biosciences (LI-COR, Inc., Lincoln, NE, USA). 3.2. NLRP3 gene knockout mice did not demonstrate
depression-like behaviors after 4-weeks CUMS
2.7. Statistical analysis
In order to evaluate the effect of CUMS on each group, the
The data were presented as mean ± standard error (SEM) and weights of mice were measured weekly, behavioral test including
differences considered statistically significant only when p-values the sucrose preference test and the tail suspension test were carried
are less than 0.05. Data was mainly analyzed using a two-way out at the end of the CUMS protocol. Generally, during the CUMS
analysis of variance (ANOVA) followed by the Bonferroni’s multi- procedure, mice in all the four groups (n = 10) gained weight week
ple comparisons post hoc test with GraphPad Prism 6 (GraphPad after week. As is presented, after two weeks’ CUMS manipulation,
Software, Inc., La Jolla, CA, USA). Two-way ANOVA for repeated the weight gaining pace of the wild-type stress group mice tended
measures was used to analyze the weight curves. Student’s t-test to slow down, while mice in the wild-type control group main-
was applied to detect significant difference between two groups, tained their increasing trend to the end of the CUMS treatment.
namely NLRP3 protein level analysis. Interestingly, both stress and unstressed group NLRP3 knockout
mice put on weight in a generally similar manner in the first three
3. Results 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
3.1. Polyacrylamide gel electrophoresis-based genotyping weight change, the wild-type mice subjected to 4 weeks’ CUMS also
analysis displayed depression-like behaviors, including decreased sucrose
preference (Fig. 2B, n = 10, *p < 0.05, F(1, 36) = 0.5476, t = 2.735) and
In order to genotype NLRP3 gene knockout mice, a poly- increased immobility time in the tail suspension test when com-
acrylamide gel electrophoresis-based method was applied. F2 pared to wild-type control group mice (Fig. 2C, n = 10, *p < 0.05, F(1,
generation homozygous NLRP3 knockout mice were adopted dur- 36) = 3.243, t = 2.343). However, the NLRP3 knockout stress group
ing the mating process of F1 generation heterozygote mice and mice didn’t demonstrate such depression-like behaviors (Fig. 2B
genotyped by T7E1 digestion method. Based on NLRP3 PCR primers 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 Considering that NF-␬B plays a pivotal role in classical inflam-
induced by NLRP3 inflammasome activation, the classical proin- matory pathway and has complicated influence on not only
flammatory cytokine IL-1␤ in both peripheral and central nervous neurogenesis but also IL-1␤ priming, we checked the phos-
system were detected respectively. As is displayed, the serumal phorylation of NF-␬B subunit p65 to represent its activation.
IL-1␤ protein levels were significantly higher in CUMS exposed Representative immunoblot bands and related statistical analyses
wild-type mice than that of the wild-type control group mice, while were displayed in Fig. 5A&B. It was suggested that phosphoryla-
the NLRP3 knockout stress group mice didn’t show an elevation of tion of p65 was significantly enhanced in wild-type CUMS mice
IL-1␤ levels (Fig. 3A, n = 8–9, **p < 0.01, F(1, 29) = 23.32, t = 3.632). than that of wild-type control mice (n = 3, **p < 0.01, F(1, 8) = 12.30,
At the meantime, the hippocampi of CUMS treated wild-type mice t = 5.504). Strikingly, NLRP3 gene knockout mice seemed to main-
contained significantly higher levels of IL-1␤ protein when com- tain p65 phosphorylation at a relatively normal level, even after
pared to wild-type control group mice, however, no such increase 4-weeks CUMS. The above results indicated that knockout of NLRP3
in hippocampal IL-1␤ protein level was detected in NLRP3 knockout inflammasome hindered NF-␬B protein activation resulted from
mice subjected to CUMS (Fig. 3B, n = 5, **p < 0.01, F(1, 16) = 6.562, CUMS exposure.
t = 4.108). Protein levels of IL-1␤ in hippocampi were normalized
to the total protein levels of each hippocampal supernatant respec-
tively. 3.6. CUMS-induced MAPK signaling pathway expression in
hippocampi

3.4. CUMS led to promoted NLRP3 expression in hippocampi Although MAPK signaling pathway has various functions upon
growth and development; it is susceptible to surrounding envi-
According to our previous studies, 4 weeks’ CUMS treatment ronment including stress and inflammation, and also affects
would result in an obvious promotion of NLRP3 protein expres- inflammatory balance in turn. Taking these circumstances into
sion. That may contribute to the following assembly and activation consideration, western blots were applied to detect the protein
of NLRP3 inflammasome. Here we detected the relative protein expression of MAPK family. Protein levels of p-JNK, p-ERK1/2,
levels of NLRP3 from each group using western blot. Apparently, and p-p38 were divided by the protein levels of JNK, ERK1/2 and
bands of wild-type CUMS group mice got stronger signal than that p38 respectively, before that they were normalized to the GAPDH
of the control group. Meanwhile, there were no visible bands in protein levels. All the other three groups’ protein levels were pre-
either stressed or non-stressed NLRP3 KO group (Fig. 4A). Densit- sented as fold changes to wild-type control group protein levels.
ometric measurements and analyses suggested that the difference The hippocampi in CUMS exposed wild-type mice had more JNK
between wild-type control group and wild-type CUMS group was and p38 phosphorylation when compared to the wild-type control
statistically significant (Fig. 4B, n = 3, *p < 0.05, t = 4.435). 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, We weighed the animals weekly to measure how their body
F(1, 8) = 5.523, t = 3.839 for p-JNK/JNK). However, the knockout of weight changed when subjected to CUMS, since weight loss could
NLRP3 inflammasome impeded CUMS-induced activation of the be one of the nine specific symptoms of MDD [1]. We found that
MAPK pathway (Fig. 6). 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
4. Discussion stress group NLRP3 knockout mice didn’t show delayed weight gain
when compared to the control group mice for the first three weeks.
In our previous studies, we found that the NLRP3 inflammasome At the end of the CUMS treatment, the weight of the stress group
signaling pathway inhibitors Ac-YVAD-CMK and VX-765 could NLRP3 knockout mice was less than the control group because two
impede LPS-induced acute depression-like behaviors and CUMS- of the mice in the stress group dropped weight sharply (Fig. 2A).
induced chronic depression-like behaviors respectively [16,17]. Unfortunately, the wild-type mice applied in this research were not
To further investigate the functional role of NLRP3 inflamma- littermate ones, we cannot definitely explain the weight variance
some in CUMS-induced depression-like behaviors and the relevant between wild-type and NLPR3 KO groups owing to possible slight
inflammatory signaling pathways, we compared the behavioral and gene background difference. However, it did not demonstrate any
molecular parameters of wild-type and NLRP3 gene knockout mice.
W.-J. Su et al. / Behavioural Brain Research 322 (2017) 1–8 7

behavioral difference between wild-type control versus NLRP3 KO ence the NF-␬B signaling pathway reversely, and NF-␬B might
control mice throughout this research. mediate the downstream inflammatory effects of NLRP3 inflam-
Sucrose preference test is widely used to assess the “anhedo- masome in depression-like behaviors.
nia” status, and tail suspension test is broadly used to indicate Extracellular signal-regulated kinase (ERK), c-Jun amino-
the behavioral despair status of animals [30,31]. In this study, the terminal kinase (JNK) and p38 proteins are members of the MAPK
results of these two tests demonstrated that after 4 weeks CUMS signal pathways. The ERKs had function in the control of cell divi-
treatment the wild-type mice displayed depression-like behaviors, sion, the JNKs are critical regulators of transcription and the p38
while the NLRP3 knockout mice were resistant to development MAPKs can be activated by inflammatory cytokines and environ-
of depression-like behaviors (Fig. 2B and C). These data further mental stressors. These proteins might be directly involved in
demonstrated that the NLRP3 inflammasome mediated CUMS- the process of neuronal cell death [40,41]. MAPKs phosphatase-1
induced depression-like behaviors. (MKP-1) was a powerful negative regulator of intracellular MAPK
Iwata et al. reported that acute restraint stress activated the signaling pathways, which were associated with depression-like
NLRP3 inflammasome and rapidly increased proinflammatory behaviors or depressive symptom severity [42–44]. However, if
cytokine IL-1␤ levels in the hippocampus [19]. Stress responses the NLRP3 inflammasome can influence the MAPK family signaling
referred to a wide array of behavioral and physiological responses, pathways remains unclear. We detected JNK, ERK and p38 proteins
and the hypothalamic-pituitary-adrenal (HPA) axis played key expression with western blot to explore if they were influenced by
roles in the integration of adaptive responses to stress [32]. Pro- the NLRP3 inflammasome. We found that the phosphorylated JNK
duction of brain IL-1 as a vital regulator of stress responses linked and p38 protein increased significantly in the hippocampus of wild-
stress-induced activation of the HPA axis and secretion of gluco- type mice after 4 weeks CUMS exposure, while the NLRP3 knockout
corticoids [33]. Goshen et al. postulated that the HPA axis underlay mice didn’t show such trends (Fig. 6). These results demonstrated
the involvement of brain IL-1␤ in stress-induced depression-like that the NLRP3 inflammasome could influence the MAPK family
behavior [11]. Koo et al. suggested that IL-1␤ mediated the antineu- stress kinases signaling pathway, and suggested that MAPK might
rogenic and depression-like behavior induced by acute and chronic mediate the downstream inflammatory effects of NLRP3 inflamma-
stress [34]. We detected serumal and hippocampal IL-1␤ levels to some in depression-like behaviors.
determine if there were NLRP3 inflammasome dependent matu- It is very regretful that in our study, we have exerted our utmost
ration and release of IL-1␤ in the peripheral and central nervous to determine the particular cell type of all the above changes in NF-
system. As we expected, the wild-type mice had significantly higher ␬B and MAPK signaling pathway but failed repeatedly. According
levels of IL-1␤ in serum and hippocampi when compared to other to new discoveries, Audrey Gustin et al. raised their opinion that
three groups. Knockout of the NLRP3 gene blocked the increase of NLRP3 inflammasome was expressed and functional in mouse brain
IL-1␤ in the peripheral and central nervous system (Fig. 3). Sim- microglia but not in astrocytes [45]. In a former research, Kreisel
ilarly, it was also confirmed that CUMS could result in promoted T et al. also indicated that disturbances in microglial functioning
NLRP3 expression in hippocampi, which was the major component has an etiological role in chronic stress-induced depression [46].
of NLRP3 inflammasome and contributed essentially to its activa- However, almost at the same time, Sonja Johann et al. claimed that
tion (Fig. 4). These data indicated that the release of mature IL-1␤ NLRP3 inflammasome was expressed by astrocytes from the spinal
was dependent on the NLRP3 inflammasome activation, which was cord in the SOD1 mouse model [47]. Apparently, the complicated
in accordance with a former research [18]. circumstances about this issue should be paid more attention and
IL-1␤ mediated CUMS-induced depression-like behavior partly need much effort in further researches, including our own in-depth
via inhibiting hippocampal neurogenesis while some antidepres- projects.
sant drugs could increase hippocampal neurogenesis [11,35].
Hippocampal neurogenesis was a critical biological and cellular
5. Conclusions
pathogenesis of MDD, and proliferation of hippocampal neural
progenitor cells could be suppressed by stress via the NF-␬B sig-
In summary, these results further demonstrated that the NLRP3
naling pathway [34]. Besides, NF-␬B mediated the priming process
inflammasome was involved in the CUMS-induced depression-like
of pro-IL-1␤ induced by priming signals such as toll-like recep-
behaviors via regulating IL-1␤ protein expression in serum and
tor agonist LPS or proinflammatory cytokine tumor necrosis factor
hippocampus. The NLRP3 inflammasome could influence the NF-
[12]. According to former researches, NF-␬B was involved in the
␬B signaling pathway and MAPK family stress kinases signaling
activation of NLRP3 inflammasome via activating pattern recogni-
pathway. These two pathways might mediate the downstream
tion receptors and regulating NLRP3 expression [36,37]. However,
inflammatory effects of NLRP3 inflammasome. Our study suggested
how the NLRP3 inflammasome might influence the NF-␬B signal-
that interventions targeting the inflammasome-IL-1␤ signaling
ing pathway remains elusive. We used immunoblot measurements
pathways, or the NF-␬B and MAPK signaling pathways might pro-
to check the activation of NF-␬B in hippocampi. After subjected to
vide promising strategies to alleviate MDD in the future.
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- Author’s contributions
ing to classical theories, transcription factors of the nuclear factor
␬ B (NF-␬B)/Rel family contained five family members in mam- W-JS and YZ (Yi Zhang) contributed equally to this work. C-
mals: RelA (p65), c-Rel, RelB, NF-␬B1 (p105/p50), and NF-␬B2 LJ and G-CL designed the research; S-JF and YZ bred the NLRP3
(p100/p52). Heterodimeric p65-p50 complex seems to be the most gene knockout mice. W-JS and YZ (Yi Zhang) conducted the estab-
abundant type of serious of homo- or heterodimeric complexes, lishment of depression mouse model; YC, HG and Y-JL assisted
also they were complexed with members of the NF-␬B inhibitor behavioral measurement and sample collection; YZ (Yi Zhang) and
(I␬B) family and held in an inactive state in the cytoplasm. Conven- YC detected IL-1␤ levels in serum and hippocampi; W-JS detected
tionally, NF-␬B was defined activated after I␬B phosphorylation MAPK pathway protein expression and NF-␬B translocation.; WP
by I␬B kinases (IKKs) and subsequently degraded, thus liberat- detected the I␬B-␣ and NLRP3 protein expression. YZ (Yi Zhang)
ing and phosphorylating the active NF-␬B complex including p65 and W-JS analyzed all the data and wrote the manuscript text with
which would translocate into the nucleus [38,39]. To date, these Y-XW, Z-LY helped with the revision of this manuscript. All authors
results suggested that the NLRP3 inflammasome could also influ- read and approved the final version of manuscript.
8 W.-J. Su et al. / Behavioural Brain Research 322 (2017) 1–8

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This study funded by the National Natural Science Foundation of IL-1beta-induced F-actin remodeling by inhibiting NF-kappaB signaling in
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