Li et al. Journal of Neuroinflammation(2021) 18:1
https://doi.org/10.1186/s12974-020-02040-8

RESEARCH

Open Access

Isoliquiritin ameliorates depression by
suppressing NLRP3-mediated pyroptosis via
miRNA-27a/SYK/NF-κB axis
Yuanjie Li1, Wen Song1, Yue Tong1, Xia Zhang1, Jianjun Zhao1, Xiaojuan Gao1, Jingjiao Yong1 andHanqing Wang1,2,3*

Abstract
Background: The NLRP3-mediated pyroptosis, which could be regulated by miRNA-27a, is a key player in thedevelopment of depression. Isoliquiritin is a phenolic flavonoid compound that has been demonstrated to suppress
NLRP3-mediated pyroptosis. However, it is still unknown whether isoliquiritin could confer antidepressant activity
via decreasing NLRP3-mediated pyroptosis by stimulating miRNA-27a. Thus, in the current study, we explored theantidepressant activity of isoliquiritin and its underlying mechanism.
Methods: Expression of miRNA-27a in depressed patients or mice was measured using qRT-PCR. Luciferase reporter
assay was performed to illustrate the link between miRNA-27a and SYK. Lipopolysaccharide (LPS) and chronic social
defeat stress (CSDS) depression models were established to investigate the antidepressant actions of isoliquiritin.
Changes in miRNA-27a/SYK/NF-κB axis and NLRP3-mediated pyroptosis were also examined. The role of miRNA-27a
in isoliquiritin-related antidepressant effect was further investigated by using miRNA-27a inhibitors and mimics ofmiRNA-27a.
(Continued on next page)* Correspondence: hqwangnx@tom.com
1
College of Pharmacy, Ningxia Medical University, 1160 Shengli Street,
Yinchuan 750004, Ningxia, People’s Republic of China
2
Ningxia Engineering and Technology Research Center for Modernization ofRegional Characteristic Traditional Chinese Medicine, Ningxia Medical
University, Yinchuan, People’s Republic of China
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which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
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Li et al. Journal of Neuroinflammation(2021) 18:1

Page 2 of 23(Continued from previous page)

Results: Our results showed the miRNA-27a expression was downregulated in the serum of depressed patients,
and decreased serum and hippocampus expression of miRNA-27a were observed in rodent models of depression.
SYK gene expression was significantly reduced by miRNA-27a mimic incubation. Isoliquiritin profoundly attenuated
LPS or CSDS-induced depressive symptoms, as well as CSDS-induced anxiety behavior. In the hippocampus, LPS
and CSDS decreased miRNA-27a mRNA expression; increased the protein levels of SYK, p-NF-κB, and NLRP3: cleaved
Caspase-1, IL-1β, and GSDMD-N: and elevated the concentration of IL-1β, IL-6, and TNF-α, which were all restoredby isoliquiritin administration. Meanwhile, isoliquiritin upregulated the hippocampal NeuN protein level, improved
the survival and morphology of neurons, and decreased pyroptosis-related neuronal cell death. Moreover,isoliquiritin protected primary microglia against LPS and adenosine triphosphate (ATP) elicited NLRP3
inflammasome activation in vitro, evidenced by declined protein levels of p-NF-κB, NLRP3; cleaved Caspase-1, IL-1β,
and GSDMD-N; upregulated miRNA-27a mRNA expression; and decreased the mRNA and protein levels of SYK.
Nevertheless, miRNA-27a inhibitors significantly reversed isoliquiritin-generated therapeutic efficacy in CSDS miceand in vitro. Furthermore, the cytoprotective effect of isoliquiritin was similar to that of miRNA-27a mimics in LPS
and ATP-treated primary microglia.
Taken together, these findings suggest that isoliquiritin possesses potent antidepressant property, which requires
miRNA-27a/SYK/NF-κB axis controlled decrease of pyroptosis via NLRP3 cascade.
Keywords: Depression, miRNA-27a, NLRP3, Pyroptosis, Isoliquiritin

Background
Depression is a pervasive and debilitating psychiatric disorder that is characterized by low mood, loss of pleasure,social avoidance, etc. It affects approximately 322 million
people worldwide, leading to a major social and economic burden [1]. Although multiple antidepressants areavailable for depression treatment, first-line drugs, including selective serotonin reuptake inhibitors (SSRIs),
exhibit low curative ratio, extensive side effects, andpoor treatment compliance. Hence, novel therapies withhigher efficacy and fewer adverse effects are urgently
needed [2].
Current evidence demonstrates that the developmentof depression is associated with NLRP3-mediated pyroptosis. Pyroptosis is a Caspase-1-dependent programmed
cell death. In contrast to apoptosis, the process of pyroptosis is proinflammatory and initiated by inflammasomes. NLRP3 is a typical representative of NOD-likereceptors (NLRs), which play essential roles in triggering
inflammasome-mediated neuroinflammation in microglia. Activated NLRP3 induces the cleavage of proCaspase-1 into activated Caspase-1, which drives thematuration of proinflammatory cytokine IL-1β andpyroptosis executor GSDMD, consequently leading topyroptosis and the production of proinflammatory factors such as IL-6 and TNF-α [3]. Clinical evidence demonstrated that the concentrations of NLRP3 activatedcytokines such as IL-1β, IL-6, and TNF-α were elevatedin the cerebral spinal fluid (CSF) and serum of patientswith depression [4]. Therapy contributing to the decrease of NLRP3-regulated pyroptosis was found to exert
significant antidepressant-like actions [5].

MicroRNAs (miRNAs) are a class of small, singlestranded, non-coding RNAs that negatively regulate target gene transcription via binding to the partial sequencehomology of the 3′-untranslated region of their target
mRNAs [6]. As one of hundreds of microRNAs,miRNA-27a has been reported to control the NLRP3
cascade via SYK signaling [7, 8]. Spleen tyrosine kinase(SYK) is a cytoplasmic protein-tyrosine kinase that expresses in either hematopoietic or epithelial cells. It is
responsible for a variety of physiological function, suchas cellular adhesion and immune recognition [9]. The
activation of miRNA-27a could downregulate SYK expression, which then stimulates NF-κB and facilitates
NLRP3 signal [10].
Isoliquiritin is one of the major flavonoid glycosidecompounds extracted from Glycyrrhiza uralensis, which
possess broad spectrum of pharmacological properties,
for instance, antiangiogenic, anti-neurotoxic, and antitumor [11–13]. Isoliquiritin has also been reported toexert antidepressant-like actions. The immobility time intail suspension test (TST) and forced swimming test
(FST) was significantly reduced after treatment withisoliquiritin, reflecting its impact on alleviating behavioral despair [14]. In addition, isoliquiritin is capable ofdeactivating NF-κB regulated inflammation response.
Decreased mRNA levels of NF-κB and IL-6 were detected in hepatocytes after isoliquiritin intervention [15].
In the kidney of membranous glomerulonephritis rats,
isoliquiritin significantly downregulated the protein
expression of NF-κB, as well as the mRNA expressions
of NF-κB and IL-1β [16]. These findings lead us tohypothesize that isoliquiritin could ameliorateLi et al. Journal of Neuroinflammation(2021) 18:1

depression via the miRNA-27a/SYK/NF-κB axismediated attenuation of pyroptosis by targeting NLRP3
pathway.
To investigate this hypothesis, first we verified the
involvement of miRNA-27a in depression using
hippocampus and serum samples obtained from depressed patients or rodent models. The relationshipbetween miRNA-27a and SYK was detected employing luciferase reporter assay. Lipopolysaccharide andchronic social defeat stress models of depression were
established to examine the impact of isoliquiritin on
depressive behaviors, hippocampal miRNA-27a/SYK/
NF-κB cascade and NLRP3-regulated pyroptosis. Finally, the role of miRNA-27a in isoliquiritin-related
antidepressant effects was further evaluated using
miRNA-27a inhibitors and mimics.

Materials and methods
Participants

Adult patients (men 12, women 12) aged 18–60 years
were enrolled at Taizhou Fourth People’s Hospital. Patients were diagnosed with MDD according to the Diagnostic and Statistical Manual of Mental Disorders, 4thed.-Text Revision (DSM-IV-TR). The severity of MDD
was assessed using the 17-item Hamilton Depression
Rating Scale (HAMD-17). Eligible patients had HDRS-17
total scores ≥ 18 at study entry and had stopped taken
anti-depressive or other psychiatric medications formore than 3 weeks. Patients pregnant or with a primary
diagnosis of schizophrenia or other psychotic disorders,
substance abuse, severe cognitive impairment, or epilepsy were excluded from the study. Healthy control
subjects (men 12, women 12) had a HAMD-17 score ≤ 7
and a Montreal Cognitive Assessment (MoCA) score ≥26 at study entry. The healthy control group consistedof individuals with no present and past history of psychiatric disorders. Written informed consent was obtained
from all participants.
AnimalMale C57BL6/J mice (4–6 weeks of age) and retired CD1 mice breeders (≥ 12 weeks of age) were obtained from
Shanghai Sipper-BK laboratory animal Co. Ltd (Shanghai, China). Mice were housed in a regulated environment (23 ± 1 °C, 60 ± 5% humidity) with a 12-h light/
12-h dark cycle (lights on at 07:00). Animals wereallowed to habituate for 1 week before the start of experiments. All studies were conducted in accordance with
the Provision and General Recommendation of Chinese
Experimental Animals Administration Legislation.
Drugs

Isoliquiritin (purity ≥ 98%) was obtained from Chengdu
Must Bio-Technology Co., Ltd (Sichuan, China).

Page 3 of 23

Fluoxetine (Flu) was purchased from Sigma-Aldrich (St.
Louis, USA). Flu (20 mg/kg) was selected as positive
control based on previous studies [17, 18].
Experimental design
Experiment I: involvement of miRNA-27a expression in
depression

qRT-PCR was performed to measure the miRNA expression in the serum and hippocampus samples of depressed patients (n = 24 per group) or mice (n = 10 pergroup). Human serum samples were obtained from depression patients and healthy donors at Taizhou Fourth
People’s Hospital. The hippocampus and serum samplesof mice experienced LPS or chronic social defeat stress(CSDS) challenge were collected.
Experiment II: effect of isoliquiritin on LPS-induced
depression

Male C57BL6/J mice were randomized into fivegroups (n = 10 per group): control group (vehicle
treatment and ICV injection of saline), LPS group(vehicle treatment and ICV injection of LPS), isoliquiritin (10 mg/kg) + LPS group, isoliquiritin (30 mg/kg) + LPS group, and fluoxetine (20 mg/kg) + LPS
group. Mice were orally administered with isoliquiritin, fluoxetine, or vehicle (0.9% saline) by gavage daily(8 a.m.) for 14 days. LPS modeling was performed on
day 11–12 (9 a.m.). On the 13th day, the sucrose preference test (SPT) (9 a.m.) and TST (3 p.m.) were carried out, and the FST (9 a.m.) was conducted on the
14th day. Subsequently (11 a.m.), mice were anesthetized with 1% pentobarbital sodium, and brains wereimmediately removed and hippocampus was collected
and stored at − 80 °C until further use.
Experiment III: effect of isoliquiritin on CSDS-induced
depression

Mice were randomly divided into five groups (n = 10 pergroup): control group (vehicle treatment), CSDS group(vehicle treatment), CSDS + isoliquiritin (10 mg/kg)
group, CSDS + isoliquiritin (30 mg/kg) group, and CSDS
+ fluoxetine (20 mg/kg) group. Mice were orally administered with isoliquiritin, fluoxetine or vehicle by gavage
daily (8 a.m.) for 14 days. The CSDS modeling was performed from at 9 a.m. from day 1 to 12. On the 13thday, the SPT (9 a.m.), open field test (OFT) (2 p.m.), andTST (5 p.m.) were carried out, and the FST (9 a.m.) andsocial interaction test (SIT) (3 p.m.) were conducted on
the 14th day. Subsequently (5 p.m.), mice were anesthetized with 1% sodium pentobarbital, and brains were immediately removed and hippocampus was collected andstored at − 80 °C until further use.

Li et al. Journal of Neuroinflammation(2021) 18:1

Experiment IV: role of miRNA-27a in isoliquiritin-related
antidepressant efficacy

Mice were randomized into five groups (n = 10 pergroup): control + LV-NC group (vehicle treatment), CSDS
+ LV-NC group (vehicle treatment), CSDS + isoliquiritin(30 mg/kg) + LV-NC group, CSDS + LV-miRNA-27a
group (vehicle treatment), and CSDS + isoliquiritin(30 mg/kg) + LVI-miRNA-27a group. Mice were orally
administered with isoliquiritin or vehicle by gavage
daily (8 a.m.) for 14 days, and lentivirus vectors(Fanyida Biosciences, Nanjing, China) expressing
miRNA-27a inhibitor (forward 5′-GGATCCAAGTGTCAGTATTCAAGGCGAATTAAGTGTCATTCAAGGCGGAATTC-3′ and reverse 5′-GAATTCCGCCTTGAATGACACTTAATTCGC CTTGAATA CTGA
CACTTGGATCC-3′) or control vectors (LV-NC) at adose of 1 × 107 transducing units each time was i.c.v.
injected 1 h prior to drug administration. The CSDS
modeling was performed from at 9 a.m. from day 1 to
12. On the 13th day, the SPT (9 a.m.) and TST (3p.m.) were carried out, and the FST (9 a.m.) and SIT(3 p.m.) were conducted on the 14th day. Subsequently (5 p.m.), mice were anesthetized with 1% sodium pentobarbital, and brains were immediately
removed and hippocampus was collected and storedat − 80 °C until further use.
Lipopolysaccharide (LPS) model

The LPS modeling was performed according to themethod reported previously [19]. Briefly, Mice wereanesthetized with pentobarbital sodium (80 mg/kg, i.p.)
and restrained onto a stereotaxic apparatus. A skin incision was made to expose the skull, and a small burr hole
was drilled perpendicularly to the skull. Using an infusion pump, 1 μl saline or 1 μl of 10 mg/ml LPS (L2630;
serotype O111:B4; Sigma) in saline was ICV administrated (flow rate, 0.3 μl/min) to mice stereotactically via
the coordinates: − 2.5 mm dorsal/ventral, − 1.0 mm lateral, and − 0.5 mm anterior/posterior from bregma. The
needle remained in place for 5 min for proper dispersalof the drug.
Chronic social defeat stress (CSDS)The CSDS paradigm was conducted as previous described with minor modifications [20]. Eight-week-old
Kunming mice were considered “intruder” mice and experienced 12 consecutive days of stress. Briefly, intruder
was defeated by a CD-1 mouse. The defeat episodelasted for 5 min, after which the defeated mouse wassubjected to continuous psychological stress from a CD1 mouse through a clear perforated divider allowing forvisual, olfactory, and auditory contact in a shared home
cage for the next 24 h.

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Behavioral studies

The behavioral tests were performed by the investigators
blinded to the study groups.
Tail suspension test (TST)The TST was performed as previously described withminor modifications [19]. The mouse with a medical
tape placed 1 cm from the tip of the tail was suspended
upside-down for 6 min on the TST instrument holder.
The immobility time for each mouse throughout the last
4 min was statistically analyzed by ANY-MAZE software.
Forced swimming test (FST)The FST was performed as previously described withminor modifications [19]. Mice were placed in a transparent cylinder (diameter 10 cm, height 30 cm), containing 20 cm of water at 24 ± 1 °C. The total duration of
immobility during the last 4 min of the 6-min session
was analyzed.
Sucrose preference test (SPT)The SPT followed a published procedure with minor
modifications [21]. Mice were accustomed to 2% sucrose
water for 3 consecutive days prior to the test. On thetesting day, each mouse was water-deprived for 24 h,
and given two drinking bottles: one containing 2% sucrose water and the other regular water. After 2 h, thebottles were weighed and sucrose preference was analyzed according the following formula: sucrose preference = sucrose intake/total water consumption × 100%.
Open field test (OFT)The OFT was applied to evaluate anxiety and locomotor
activity in rodents as previously described [21]. Animals
were gently placed into open-field chambers (30 × 30cm) which were equipped with video cameras. During
the test, mice were allowed explore the arena for 5 min.
Total distance traveled and time spent in the central
area automatically were recorded.
Social interaction test (SIT)

The social interaction test was used to determine social
avoidance behavior [22]. Briefly, the mouse was introduced to an open field chamber where a male CD-1
mouse was present in the mesh cage at one end. Eachmouse was allowed to freely explore the environmentfor 2.5 min with its movement tracked. The mouse was
habituated to the chamber in the absence of a CD-1
mouse for 2.5 min prior to the test. Social interaction ratio was calculated according the following formula: Social interaction ratio = time spent in the interaction
zone in the presence of target/time spent in the interaction zone in the absence of target × 100%.

Li et al. Journal of Neuroinflammation(2021) 18:1

Cell culture and drug treatments

Mouse primary microglia were obtained from the hippocampus of 1-day-old neonatal C57/B6J mice following apublished protocol [23]. Briefly, hippocampal tissues
were dissected and digested in 0.125% Trypsin-EDTA
for 15 min at 37 °C, followed by mechanical shearing.
After centrifugation (1000 rpm, 10 min), cells were resuspended in Dulbecco’s modified Eagle medium(DMEM) with additional 10% FBS. Then, the resulting
suspension was filtered through a 70-μm filter and cultivated in DMEM supplemented with 10% FBS, 40 U/mL
penicillin, and 40 μg/mL streptomycin. Medium wasrefreshed every 3–4 days. Upon reaching confluence(10–12 days), the microglial cells were separated fromthe underlying astrocytic monolayer via shaking off for
5 h at 100 rpm. The cells were verified by Iba-1 immunostaining. After 12 h of culture, the microglial cells wereready for use.
Microglial cells were exposed to different concentrations of isoliquiritin for 2 h, and then added with LPS(10 μg/L) and ATP (5 mM) for 12 h to induce NLRP3
inflammasome activation phenotypes [5].
Quantitative real-time polymerase chain reaction (qRTPCR) analysis

Total RNA in hippocampus, serum, and cells were extracted with TRIzol reagent (Invitrogen USA) and reverse transcribed with a first strand cDNA synthesis kit(Transgen Biotech, China) following the manufacturer’s
guides. GAPDH was used as normalization control formRNA. The sequences for primers were as follows:

Page 5 of 23

measured using the Bradford assay kit. Proteins wereseparated on 10% SDS-PAGE gels and then transferred
to polyvinylidene difluoride (PVDF) membranes. After
blocking with 3% BSA, the membranes were incubatedwith primary antibodies at 4 °C overnight. Then, appropriate HRP-conjugated secondary antibodies were added
and incubated for 1.5 h at room temperature. The blots
were visualized using ECL western blotting detection reagents and analyzed by Image J software. Antibodies
were obtained from Cell Signal Technology or Santa
Cruz Biotechnology.
Enzyme-linked immunosorbent assay (ELISA)

After behavioral test, hippocampus or serum was harvest
from mice. The concentrations of IL-1β, IL-6, and TNFα were quantified using ELISA kits (Boster BiologicalTechnology, Wuhan, China) according to the manufacturer’s instructions.
Nissl staining

Nissl staining was carried out using Nissl Staining
Solution (Beyotime Institute of Biotechnology, Shanghai,
China) in accordance with the manufacturer’s protocols.
Nissl-positive cells were viewed under an optical microscope (Olympus, Japan).
TUNEL staining

Pyroptosis-related cell death was detected by TUNEL
staining using a kit according to the manufacturer's
instruction (Roche, South San Francisco, CA, USA).
MTT assay

Primers Forward

ReversemiR-27a 5′-TTCACAGTGGCTAAG-3′5′-GTGCAGGGTCCGAGGT-3′

SYK

5′-CCAACCACCTGACCTACT
TTTT-3′5′-ATTAAGTTCCCTCTCGAT
GGTG-3′GAPDH

5′-ATGGAGAAGGCTGGGGCTC- 5′-AAGTTGTCATGGATGACC
3′
TTG-3

Luciferase reporter assay

The luciferase reporter assay was performed aspreviously reported [7]. The HZ0471 luciferase reporter
plasmid containing the wild-type (WT) or mutant(MUT) 3′UTR fragment of SYK was co-transfected into
HEK293T cells co-transfected with miRNA-27a. After48 h of cell transfection, the luciferase reporter gene activity was measured using Glomax 2020 luminometer.

The cytotoxicity was analyzed using MTT assay as
previously described [24]. The viability of living cells wascalculated as percentage of control.
Immunocytochemistry

The expression of Iba-1 was determined by immunocytochemistry as previously described [23]. Primary
microglia cells were fixed with 4% paraformaldehyde
for 20 min and then permeabilized with 0.2% Triton
X-100 for another 15 min. After blocking with 10%
BSA, immunostaining was performed by incubatingwith rabbit-anti-Iba-1 (2.5ug/ml; Wako 019-19741)
overnight at 4 °C followed by goat anti-rabbit IgG
H&L (Alexa Fluor® 488, 1: 200) for 2 h at room
temperature. The nuclei were stained with DAPI for
10 min. The fluorescent images were examined undera confocal microscope.

Western blot

The Western blot followed a published procedure withminor modifications [23]. Proteins were extracted fromthe hippocampus of LPS and CSDS mice, and thetreated cells, and total protein concentrations were

Cell transfection

MiR-27a mimics and inhibitors were transfected withlipofectamine2000 (Life Technologies) following the
manufacturer’s protocols. Before transfection, cells wereLi et al. Journal of Neuroinflammation(2021) 18:1

cultured in 6-well plates till they reached 80% confluency. Then, 20 nM miR-27a mimics (same operation
for miR-27a inhibitors) were transfected into cells at
37 °C for 24 h using Lipofectamine® 2000 (Invitrogen).
miR-27a mimics (5′-CGCCUUGAAUCGGUGACA
CUU-3′) and miR-27a inhibitors (5′-UGGACAUUUUUAAAAACUGUGAU-3′) were obtained from General
Biol Co., Ltd. (Anhui, China).
Statistical analysis

All data are presented as mean ± SEM. Data wereanalyzed by unpaired Student’s t test for comparison
between two groups. Data were analyzed by one-way or
two-way ANOVA with Tukey post hoc test for comparison between multiple groups. The criterion for significance was p < 0.05.

Results
Serum miRNA-27a level is upregulated in MDD patients

To identify the change in miRNA-27a mRNA expression associated with depression, we detected themiRNA-27a mRNA concentration in the serum ofdepressed patients (Fig. 1a). The qRT-PCR results
demonstrated that compared with normal subjects,
the miRNA-27a mRNA expression was significantly
lower in the serum of depressed patients (p =0.0469). This result indicated that the reduction of
miRNA-27a expression was closely associated withthe development of depression.
Serum and hippocampal miRNA-27a expressions were
elevated in rodent models of depression

Next, we examined the miRNA-27a expression in rodent
models of depression. As illustrated in Fig. 1b and c,
LPS (F (1, 36) = 68.21, p < 0.0001) or CSDS (F (1, 36) =94.51, p < 0.0001) challenged mice exhibited decreased
miRNA-27a mRNA level in the serum and hippocampus

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relative to their control counterparts (p < 0.05), supporting the reduction of miRNA-27a expression in depression pathology.
SYK is a target gene of miRNA-27a

The binding relationship between miRNA-27a and SYK
was predicted using online analysis tool TargetScan andverified by employing luciferase reporter assay, in which
the 3′-UTR of SYK was fused with a gene encoding
luciferase (Fig. 2). The results showed that the relative
luciferase activity of HEK293T cells transfected withSYK-WT was notably suppressed by miRNA-27a mimic(F (1, 12) = 5.773, p = 0.0334), suggesting that SYKserves as a potential target of miRNA-27a.
Isoliquiritin ameliorated LPS-induced depression
Isoliquiritin attenuated LPS-induced depressive behaviors

We examined whether isoliquiritin treatment could
alleviate LPS-induced depressive behaviors in mice
(Fig. 3a). Exposure to LPS significantly reduced thesucrose preference ratio (F (4, 45) = 29.92, p <0.0001) during the SPT assessment compared with
control mice (Fig. 3d). Furthermore, LPS challenge
increased the immobility duration in TST (F (4, 35) =25.05, p < 0.0001) and FST (F (4, 45) = 35.70, p <0.0001) tasks (Fig. 3b, c). Nonetheless, isoliquiritinintervention (10 mg/kg; 30 mg/kg) and Flu completely
reversed LPS-induced reduction in sucrose preference
and suppressed the immobility time in TST and FST
tests. These results indicated that isoliquiritin and Flu
were capable of mitigating depressive symptoms in
LPS-challenged mice.
Isoliquiritin reversed miRNA-27a/SYK/NF-κB cascade

The mRNA level of miRNA-27a in hippocampus wasmeasured by qRT-PCR (Fig. 4a). Compared with thecontrol samples, the miRNA-27a expression wasFig. 1 miRNA-27a was downregulated in depression. a miRNA-27a mRNA expression in the serum of depressed patients (n = 24 per group). b
miRNA-27a mRNA level in the serum or hippocampus of LPS mice (n = 10 per group). c miRNA-27a mRNA level in the serum or hippocampus of
CDSD mice (n = 10 per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, compared to normal control groupLi et al. Journal of Neuroinflammation(2021) 18:1

Page 7 of 23

Fig. 2 SYK is a target gene of miRNA-27a. a miRNA-27a and SYK binding site prediction. b Luciferase activity in luciferase reporter gene assay.
Data are presented as mean ± SEM (n = 4 per group). *p < 0.05, compared to NC miR in WT group

Fig. 3 Effects of isoliquiritin on LPS-induced depressive behaviors. a Schematic timeline of the experimental procedure. b Sucrose preference test.
c Tail suspension test. d Forced swimming test. Data are presented as mean ± SEM (n = 8–10 per group). ##p < 0.01, compared to control group;**p < 0.01, compared to LPS groupLi et al. Journal of Neuroinflammation(2021) 18:1

Page 8 of 23

Fig. 4 Effects of isoliquiritin on miRNA-27a mRNA expression and protein levels of NLRP3 cascade in the hippocampus of LPS mice. a mRNA
expression level of miRNA-27a. b Representative Western blots. c–h Protein levels of SYK, p-NF-κB, NLRP3, cleaved Caspase-1, IL-1β, and GSDMDN. Data are presented as mean ± SEM (n = 3–4 per group). ##p < 0.01, compared to control group; *p < 0.05, **p < 0.01, compared to LPS group

significantly decreased in the hippocampus of LPS mice(F (3, 12) = 15.97, p = 0.0002). While markedly enhanced miRNA-27a level was observed in LPS mice thatreceived isoliquiritin treatment (10 mg/kg; 30 mg/kg) incomparison to LPS model group. The Western blot
assay revealed that LPS modeling elevated SYK (F (3, 8)= 16.04, p = 0.0010) and p-NF-κB (F (3, 8) = 10.22, p =0.0041) protein levels (Fig. 4b–d). However, isoliquiritinintervention had a significant effect on rescuing theexpression of SYK (10 mg/kg; 30 mg/kg) and p-NF-κB(30 mg/kg). Collectively, these findings reflected that isoliquiritin administration was effective on rescuing LPSinduced abnormality in the miRNA-27a/SYK/NF-κB
cascade.
Isoliquiritin prevented LPS-induced inflammation response

Since the NLRP3 inflammasome signaling is a potential
target of NF-κB and serves as an important role inLi et al. Journal of Neuroinflammation(2021) 18:1

depression, the expression and concentration of the
NLRP3 cascade were detected by Western blot and
ELISA separately. As presented in Fig. 4e–h, the expression profile of NLRP3 cascade including NLRP3 (F (3, 8)= 13.59, p = 0.0017), cleaved Caspase-1(F (3, 8) = 11.69,
p = 0.0027), IL-1β (F (3, 8) = 25.75, p = 0.0002), and
GSDMD-N (F (3, 8) = 104.4, p < 0.0001) were dramatically increased following LPS stimulation, which were allreversed by isoliquiritin 30 mg/kg. In addition, isoliquiritin 10 mg/kg successfully inhibited the upregulation of
IL-1β and GSDMD-N. In line with these results, increased serum levels of IL-1β (F (3, 28) = 30.24, p <0.0001), IL-6 (F (3, 28) = 156.6, p < 0.0001), and TNF-α(F (3, 32) = 77.39, p < 0.0001) induced by LPS challenge
were remarkably suppressed by the two dosages of isoliquiritin (Fig. 5).
Isoliquiritin affected neuronal cell death

In order to investigate the impact of isoliquiritin on
neuronal cell death resulted from pyroptotic cascades,
we observed the survival and alternation of neuronalcells, DNA fragmentation and expression level of NeuN
using Nissl staining, TUNEL assessment, or Western
blot approach.
Nissl staining revealed that the neurons in the
hippocampus of control mice were clear and intact.
Whereas, LPS mice exhibited reduced survived neurons
and increased impaired neurons (F (3, 8) = 11.47, p =0.0029), which had irregular neuronal cell bodies,
shrinking and hyperchromatic nuclei (Fig. 6a, b). After
isoliquiritin administration, the degree of Nissl body loss
(30 mg/kg) was attenuated, and the disorderedarrangement of neuronal cells was improved.
In TUNEL assay, we observed elevated TUNELpositive cells in the hippocampus of LPS-challenged
mice compared to control group (F (3, 8) = 168.2, p

Page 9 of 23< 0.0001), which was successfully suppressed by 10
mg/kg or 30 mg/kg of isoliquiritin administration (Fig.
6c, d).
Similar result was observed in Fig. 6e, wherein we
present the protein level of NeuN (F (3, 8) = 14.93, p =0.0012). Evident enhancement of NeuN protein level
was found in isoliquiritin group (30 mg/kg) relative to
the LPS group.
Taken together, these findings demonstrated thatisoliquiritin protect LPS mice against pyroptotic cascadecaused neuronal cell death.

Isoliquiritin ameliorated CSDS-induced depression
Isoliquiritin improved CSDS-induced depression- andanxiety-related behaviors

Depression-like behaviors were examined via multiple
behavioral tests, namely SPT, TST, FST, and SIT (Fig.
7a). The model group demonstrated decreased
sucrose preference rate in SPT (F (4, 35) = 30.97, p <0.0001) as well as extended immobility in TST (F (4,35) = 26.28, p < 0.0001) and FST (F (4, 35) = 39.49,
p < 0.0001) tasks when compared with those in thecontrol group (Fig. 7b–d). Treatment with isoliquiritin(10 mg/kg; 30 mg/kg) and Flu significantly increased
the sucrose consumption and decreased theimmobility time in TST and FST. In addition, after
repeated social defeat stress, defeated mice displayed
reduced social interaction ratio compared withundefeated controls (F (4, 35) = 13.29, p < 0.0001),
suggesting a development of social avoidance (Fig. 7g,h). Nonetheless, isoliquiritin (30 mg/kg) and Flu
administration promoted the social interaction ratio.
These findings reflected that isoliquiritin could
improve depression-like behaviors in CSDS-induced
mice.

Fig. 5 Effects of isoliquiritin on concentration of IL-1β, IL-6, and TNF-α in the hippocampus of LPS mice. a IL-1β, b IL-6, and c TNF-α. Data arepresented as mean ± SEM (n = 8–9 per group). ##p < 0.01, compared to control group; **p < 0.01, compared to LPS groupLi et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 6 Influence of isoliquiritin on pyroptosis-related neuronal death in the hippocampus of LPS mice. a, b Nissl staining of survived neurons. c, d
TUNEL assay of neuron death. e NeuN protein expression in Western blot. Data are presented as mean ± SEM (n = 3 per group). ##p < 0.01,
compared to control group; *p < 0.05, **p < 0.01, compared to LPS group

OFT was applied to assess locomotor activity andanxiety-like behavior. As presented in Fig. 7e and f,no significant difference was detected in total distance, indicating no alternation of locomotor activity
after CSDS modeling or drug treatment. However,
CSDS mice exhibited noticeable anxiety-like behavior, as indicated by less time spent in center zone
relative to the controls, while this reduction wascompletely reversed by the administration of isoliquiritin (30 mg/kg) but not Flu, demonstrating thatisoliquiritin was efficacious on ameliorating CSDSelicited anxiety.

Isoliquiritin stimulated miRNA-27a/SYK/NF-κB cascade

The efficacy of isoliquiritin on the miRNA-27a/SYK/
NF-κB cascade in CSDS mice was shown in Fig. 8a–d. After CSDS, the mRNA expression of miRNA-27a(F (3, 8) = 79.43, p < 0.0001) was decreased in the
hippocampus, along with increased protein levels of
SYK (F (3, 8) = 8.720, p = 0.0067) and p-NF-κB (F(3, 8) = 14.24, p = 0.0014) compared to the control.
However, isoliquiritin significantly upregulated themiRNA-27a mRNA level (10 mg/kg; 30 mg/kg) anddownregulated the protein expression of SYK (30 mg/kg) and NF-κB (30 mg/kg), indicating that isoliquiritinLi et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 7 Effects of isoliquiritin on CSDS-induced depression- and anxiety-related behaviors. a Schematic timeline of the experimental procedure, b
sucrose preference test, c tail suspension test, d forced swimming test, e, f open field test, and g, h social interaction test. Data are presented asmean ± SEM (n = 8 per group). #p < 0.05, ##p < 0.01, compared to control group; *p < 0.05, **p < 0.01, compared to CSDS groupLi et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 8 Effects of isoliquiritin on miRNA-27a mRNA expression and protein levels of NLRP3 cascade in the hippocampus of CSDS mice. a mRNA
expression level of miRNA-27a. b Representative Western blots. c–h Protein levels of SYK, p-NF-κB, NLRP3, cleaved Caspase-1, IL-1β, and GSDMDN. Data are presented as mean ± SEM (n = 3 per group). ##p < 0.01, compared to control group; *p < 0.05, **p < 0.01, compared to CSDS group

alleviated miRNA-27a/SYK/NF-κB cascade defects in
CSDS mice.
Isoliquiritin reduced CSDS-induced inflammation response

We next evaluated the effect of isoliquiritin on
inflammation response applying Western blot and
ELISA assays. As shown in Fig. 8e–h, chronical social
defeat stress resulted in elevated protein levels of
NLRP3 (F (3, 8) = 51.87, p < 0.0001), cleaved Caspase-1(F (3, 8) = 21.34, p = 0.0004), IL-1β (F (3, 8) = 48.51, p< 0.0001), and GSDMD-N (F (3, 8) = 35.95, p < 0.0001)
in Western blot assessment. Increased serum concentration of IL-1β (F (3, 28) = 108.4, p < 0.0001), IL-6 (F(3, 28) = 138.0, p < 0.0001) and TNF-α (F (3, 28) =49.49, p < 0.0001) were also found in CSDS-challenged
mice (Fig. 9), while these effects were completely reversed by isoliquiritin 30 mg/kg and partially by isoliquiritin 10 mg/kg, suggesting that treatment with theisoliquiritin significantly ameliorated inflammation response in CSDS mice.

Li et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 9 Effects of isoliquiritin on concentration of IL-1β, IL-6, and TNF-α in the hippocampus of CSDS mice. a IL-1β, b IL-6, and c TNF-α. Data arepresented as mean ± SEM (n = 8 per group). ##p < 0.01, compared to control group; **p < 0.01, compared to LPS group

Isoliquiritin affected neuron death

We detected the survival and alternation of neuronalcells, DNA fragmentation and NeuN expression todetermine if pyroptotic induced cell death was blocked
by isoliquiritin administration.
In Nissl staining, the neurons in the hippocampus
were clear and intact in control group (Fig. 10a, b).
Whereas in CSDS model group, the number of Nisslpositive cells was noticeably decreased (F (3, 8) =17.58, p = 0.0007), and impaired neurons increased,
which had irregular neuronal cell bodies, shrinking
and hyperchromatic nuclei. Interestingly, these effects
were clearly mitigated by isoliquiritin intervention (30mg/kg).
The results in TUNEL staining demonstrated thatthe model group exhibited an evident increase in
TUNEL-positive cells (F (3, 8) = 127.2, p < 0.0001)
compared with the control group (Fig. 10c, d). However, the apoptotic cell number markedly declined
after treatment with isoliquiritin (10 mg/kg; 30 mg/kg).
The protein level of NeuN was analyzed using
Western blot (Fig. 10e). Declined NeuN level wasobserved in CSDS mice (F (3, 8) = 33.57, p < 0.0001),
which was restored by isoliquiritin treatment (10 mg/kg;
30 mg/kg).
miRNA-27a inhibitor compromised isoliquiritin-generated
therapeutic efficacy in CSDS mice
miRNA-27a inhibitor blocked isoliquiritin-generated efficacy
on depressive behaviors

To elucidate the mechanism of miRNA-27a in regulating
isoliquiritin-related antidepressant efficacy, lentivirus
miRNA-27a inhibitor was used in this study (Fig. 11a).
Clearly, treatment with isoliquiritin (30 mg/kg) increased
the sucrose preference in SPT (F (1, 36) = 22.38, p <0.0001) and social interaction in SIT (F (1, 28) = 18.85, p= 0.0002), and shortened the immobility duration inboth TST (F (1, 36) = 26.42, p < 0.0001) and FST (F (1,36) = 25.70, p < 0.0001) tests on CSDS model (Fig. 11b–f). However, pretreatment with miRNA-27a inhibitor
significantly reversed isoliquiritin-generated beneficial
effects in SPT (F (1, 36) = 11.61, p = 0.0016), SIT (F (1,28) = 8.193, p = 0.0079), TST (F (1, 36) = 6.083, p =0.0185), and FST (F (1, 36) = 14.35, p = 0.0006) assessments. These data demonstrated that activation of
miRNA-27a is required for the antidepressant actions ofisoliquiritin.
miRNA-27a inhibitor abolished isoliquiritin-generated
effects on miRNA-27a-regulated pyroptosis signaling

We also explored whether miRNA-27a is required for
isoliquiritin-generated stimulation of miRNA-27aregulated pyroptosis pathway in the hippocampus (Fig.
12). In the hippocampus of CSDS mice, treatment withisoliquiritin (30 mg/kg) elicited a significant promotionin the mRNA expression of miRNA-27a (F (1, 8) =47.11, p = 0.0001), and obvious decrease in the protein
levels of SYK (F (1, 8) = 25.72, p = 0.0010), p-NF-κB (F(1, 8) = 13.09, p = 0.0068), and pyroptosis executor
GSDMD-N, which were all ablated by miRNA-27a inhibitor (miRNA-27a, F (1, 8) = 30.26, p = 0.0006; SYK, F(1, 8) = 29.75, p = 0.0006; p-NF-κB, F (1, 8) = 20.48, p =0.0019). These results support the conclusion thatmiRNA-27a activation is necessary for isoliquiritininduced restoration in miRNA-27a mediated pyroptosis
pathway.
miRNA-27a inhibitor compromised isoliquiritin-generated
effects on pyroptosis-related neuron deathTo further investigate the impact of miRNA-27a on
isoliquiritin-induced attenuation of pyroptosis-relatedLi et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 10 Influence of isoliquiritin on pyroptosis-related neuronal death in the hippocampus of CSDS mice. a, b Nissl staining of survived neurons.
c, d TUNEL assay of neuron death. e NeuN protein expression in Western blot. Data are presented as mean ± SEM (n = 3 per group). ##p < 0.01,
compared to control group; *p < 0.05, **p < 0.01, compared to LPS groupneuron death, Nissl staining, TUNEL assay, and Western
blot were carried out (Fig. 13). Nissl staining and TUNEL
assay results indicated that isoliquiritin (30 mg/kg) improved the survival and morphology of neurons (F (1, 8) =13.37, p = 0.0064), as well as pyroptosis-induced cell death(F (1, 8) = 62.35, p < 0.0001) in CSDS mice. In Western
blot assay, the upregulated NeuN Protein expression wasobserved in CSDS mice after isoliquiritin treatment (30mg/kg). Nonetheless, these effects were all abolished bymiRNA-27a inhibitor (Nissl, F (1, 8) = 20.90, p = 0.0018;

TUNEL, F (1, 8) = 39.17, p = 0.0002). These findings suggested that isoliquiritin-related efficacy on pyroptosis wasdependent on miRNA-27a activation.
Role of miRNA-27a in the effect of isoliquiritin in
alleviating LPS and ATP induced neuroinflammationin vitro

Next, we examined the effect of isoliquiritin on NLRP3
inflammasome activation in vitro by stimulating primary
microglia cells with LPS and ATP (Fig. 14a).

Li et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 11 Impact of miRNA-27a inhibitor on isoliquiritin-generated antidepressant and anxiolytic efficacy in CSDS mice. a Schematic timeline of theexperimental procedure. b Sucrose preference test (n = 13 per group). c Tail suspension test (n = 10 per group). d Forced swimming test (n = 10
per group). e, f Social interaction test (n = 8 per group). Data are presented as mean ± SEM. ##p < 0.01, compared to blank group; **p < 0.01,
compared to CSDS group; &p < 0.05, &&p < 0.01, compared to CSDS+ isoliquiritin group

Preliminary experiment was performed in order tomeasure the toxicity of isoliquiritin using MTT assay(Fig. 14b). The result showed that the maximum
concentration that had no effect on reducing cell
viability was 50 μM for isoliquiritin (F (5, 30) = 20.21, p< 0.0001). Thus, this dose was used for future
experiments.
Our data suggested that LPS and ATP treatment
significantly decreased miRNA-27a mRNA expression
and elevated SYK mRNA level, and upregulated the protein expressions of SYK, p-NF-κB, NLRP3, cleaved
Caspase-1, IL-1β, and GSDMD-N in primary microglia(p < 0.05, Fig. 15). Nonetheless, pretreatment withisoliquiritin rescued the abnormality in miRNA-27a (F(1, 8) = 39.67, p = 0.0002) and SYK (F (1, 8) = 18.84, p =0.0025) mRNA levels, and restored the proteins levels of
SYK (F (1, 8) = 54.28, p < 0.0001), p-NF-κB (F (1, 8) =20.92, p = 0.0018), NLRP3 (F (1, 8) = 41.58, p = 0.0002),
cleaved Caspase-1 (F (1, 8) = 34.44, p = 0.0004), IL-1β (F(1, 8) = 68.30, p < 0.0001), and GSDMD-N (F (1, 8) =63.86, p < 0.0001). Interestingly, the protective effects ofisoliquiritin on miRNA-27a (F (1, 8) = 26.34, p = 0.0009)
and SYK (F (1, 8) = 25.92, p = 0.0009) mRNA expressions as well as protein levels of SYK (F (1, 8) = 17.84, p= 0.0029), p-NF-κB (F (1, 8) = 15.38, p = 0.0044), NLRP3(F (1, 8) = 33.34, p = 0.0004), cleaved Caspase-1(F (1, 8)

Li et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 12 Impact of miRNA-27a inhibitor on isoliquiritin-generated deactivation of pyroptosis signaling in the hippocampus of CSDS mice. a mRNA
expression level of miRNA-27a. b Representative Western blots. c–e Protein levels of SYK, p-NF-κB, and GSDMD-N. Data are presented as mean ±SEM (n = 3 per group). ##p < 0.01, compared to blank group; *p < 0.05, **p < 0.01, compared to CSDS group; &p < 0.05, &&p < 0.01, compared to
CSDS+ isoliquiritin group= 19.94, p = 0.0021), IL-1β (F (1, 8) = 36.15, p = 0.0003),
and GSDMD-N (F (1, 8) = 22.50, p = 0.0015) were all reversed by miRNA-27a inhibitor.
Moreover, Fig. 16 revealed that isoliquiritin
ameliorated LPS and ATP induced decline in miRNA27a mRNA expression (F (1, 8) = 50.92, p < 0.0001)
and increase in proteins levels of SYK (F (1, 8) =24.70, p = 0.0011), p-NF-κB (F (1, 8) = 34.79, p =0.0004), NLRP3(F (1, 8) = 39.02, p = 0.0002), cleaved
Caspase-1 (F (1, 8) = 69.14, p < 0.0001), IL-1β (F (1,8) = 38.29, p = 0.0003), and GSDMD-N (F (1, 8) =50.13, p = 0.0001), which was similar to the effects
elicited by miRNA-27a mimics.

Discussion
In this study, we explored the antidepressant property
of isoliquiritin and its potential mechanism (Fig. 17).
Our results showed that isoliquiritin successfully
attenuated LPS- or CSDS-induced depressive symptoms and CSDS-elicited anxiety behaviors. In the
hippocampus, expression levels of miRNA-27a/SYK/
NF-κB cascade and NLRP3-mediated inflammation response and pyroptosis were all improved after isoliquiritin administration. Furthermore, isoliquiritin

protected primary microglia against LPS and ATP induced NLRP3 inflammasome activation in vitro, evidenced by declined protein levels of p-NF-κB, NLRP3,
cleaved Caspase-1, IL-1β, and GSDMD-N, promoted
miRNA-27a mRNA expression and reduced themRNA and protein levels of SYK. However, miRNA27a inhibitor significantly reversed isoliquiritingenerated therapeutic efficacy in CSDS mice andin vitro. Moreover, the cytoprotective effect of isoliquiritin was similar to that of miRNA-27a mimics in
LPS and ATP-treated primary microglia.
LPS depression model is widely used to study
inflammation-associated depression. Lipopolysaccharide(LPS) is a component of the cell membrane of Gramnegative bacteria, which provokes the production of proinflammatory cytokines in brain and periphery [25]. In
rodents, LPS triggered the development of sickness behaviors such as decrease in body weight, food intake,and locomotor activity that resolves after 14–18 h,followed by a phase of depression-like behaviors including declined sucrose preference rate and prolonged immobility duration in TST and FST [26, 27].
Chronic social defeat stress (CSDS) paradigm has
been widely used to explore the mechanisms

Li et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 13 Influence of miRNA-27a inhibitor on isoliquiritin-generated decrease of neuronal death in the hippocampus of CSDS mice. a, b Nissl
staining of survived neurons. c, d TUNEL assay of neuron death. e NeuN protein expression in Western blot. Data are presented as mean ± SEM(n = 3 per group). ##p < 0.01, compared to blank group; **p < 0.01, compared to CSDS group; &&p < 0.01, compared to CSDS+ isoliquiritin group

underlying the pathogenesis of depression- andanxiety-related behaviors [28]. In real-life situations,people frequently encounter stimuli resulted fromthe interaction with other people, and social challenge appears to be one of the most prevalentstressors in humans and social animals [29]. Repeated exposures to social defeat stress in rodents
result in significant anhedonia, behavioral despair,social avoidance, and heightened anxiety [30]. In ourstudy, mice experienced LPS or CSDS displayed decreased sucrose preference rate and social interaction
ratio, and prolonged immobility in TST and FST,

suggesting the development of depression after LPS
or CSDS challenges. However, isoliquiritin administration profoundly improved the behavior defects in
SPT, TST, FST, and SIT, indicating that isoliquiritin
was effective in alleviating depression in mice. In
previous study, isoliquiritin was shown to mitigate
behavioral despair in normal mice [14]. Here, we
further demonstrated that isoliquiritin could confer
antidepressant-like activity in the rodent models ofdepression. Additionally, existing evidence suggested
that CSDS mice displayed heightened anxiety in
OFT test, which is consistent with our result [31].

Li et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 14 Details of primary mouse microglial cell culture and cell viability following isoliquiritin treatment. a Iba-1 immunostaining images ofprimary microglia isolated using shaking. Scale bar = 50 μm. b Cell viability in MTT assay. Data are presented as mean ± SEM (n = 6 per group).
**p < 0.01, compared to control group

Nonetheless, treatment with isoliquiritin evidently increased time spent in center in OFT, indicative of ananxiolytic effect of isoliquiritin.
NLRP3 is one of the most extensively studied
NOD-like receptors, which play essential roles intriggering inflammasome-mediated neuroinflammation in microglia. The activation of NLRP3 promotes the secretion of Caspase-1, which increases
the cleavage of IL-1β and GSDMD, and then induces pyroptosis and inflammation response. The
NLRP3 cascade has been implicated in multiple diseases, such as Alzheimer’s disease, Parkinson’s disease, and stroke. Gene expression analysis revealed
that the expression levels of NLRP3, Caspase-1, and
IL-1β were elevated in the cultured PBMCs from AD
patients [32]. Enhanced IL-1β expression andCaspase-1 activity were detected in the serum samplesof PD patients, and increased levels of NLRP3,
Caspase-1, and IL-1β were found in the midbrain of
PD mice model [33]. NLRP3-inflammasome inhibitor
MCC950 suppressed the activation of Caspase-1 andIL-1β, leading to reduction of infarction, edema, andHb content as well as improved neurological deficits
in transient middle cerebral artery occlusion micemodel [34].
The NLRP3 cascade is also involved in depression.
Increased NLRP3 and IL-1β activity were observed in
the prefrontal cortex of depressed rats [35]. Melatonin, a hormone produced from L-tryptophan, remarkably enhanced LPS-induced depressive symptoms
in FST and TST assessments, which was linked to thedeactivation of NLRP3 controlled inflammation
response and pyroptosis [5]. Postmortem studies demonstrated that the protein expression and mRNAlevels of IL-1β, IL-6, and TNF-α were remarkably increased in the prefrontal cortex of depressed subjects
who died by suicide [36]. In our study, mice underwent LPS or CSDS challenge displayed elevated levels
of NLRP3, Caspase-1, IL-1β, IL-6, GSDMD, and TNFα; decreased neuron survival; and increased neuronalcell death, which were all attenuated by isoliquiritinintervention. These results confirming the essential

Li et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 15 Effect of miRNA-27a inhibitor on isoliquiritin-generated cytoprotective effects in LPS+ATP-treated primary microglia cells. a, b mRNAlevels of miRNA-27a and SYK. c Representative Western blots. d–i Protein expressions of SYK, p-NF-κB, NLRP3, cleaved Caspase-1, IL-1β, and GSDM
D-N. Data are presented as mean ± SEM (n = 3 per group). ##p < 0.01, compared to blank group; *p < 0.05, **p < 0.01, compared to LPS+ATP
group; &p < 0.05, &&p < 0.01, compared to LPS+ATP+Isoliquiritin grouprole of NLRP3-mediated pyroptosis cascade in thedevelopment of depression and Isoliquiritin-related
effects on depressive behaviors.
miRNA-27a is a member of the miRNA family,
which modulates gene expression of various biologicalprocesses including immune response [37]. miRNA27a was documented to directly target SYK and thenstimulate the NF-κB signal [7, 8]. In this study, theexpression of the SYK gene was significantly decreased after miRNA-27a mimic incubation, confirming that regulation function of miRNA-27a on SYK.
The miRNA-27a/SYK/NF-κB axis has been proposed
to regulate the NLRP3 cascade. For instance, overexpression of miRNA-27a was able to suppress IL-1βinduced inflammatory response in chondrocytes [38].
SYK inhibitor R406 significantly deactivated NLRP3,

Caspase-1, and GSDMD-N signaling; reduced theconcentration of IL-1β; and attenuated GSDMD-N induced membrane pores in mice with ischemic stroke[39]. Ju et al. discovered that LPS treatment obviously
increased the levels of proinflammatory cytokines including TNF-α, IL-1β, and IL-6 in the BAL fluid ofmice that received LPS injection, and promoted thenumber of TUNEL-positive cells in the lung, whileagomir-27a intervention greatly alleviated LPSinduced increase of proinflammatory cytokines andcell death [40].
The miRNA-27a/SYK/NF-κB axis was also involved
in depression. Postmortem brain studies revealed thatthe miRNA-27a expression was downregulated in theprefrontal cortex of depressed suicide subject [41].
Depressed rats exhibited decreased levels of miRNA-Li et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 16 Effect of miRNA-27a mimics and isoliquiritin in LPS+ATP-treated primary microglia cells. a mRNA expression of miRNA-27a. b
Representative Western blots. c–h Protein levels of SYK, p-NF-κB, NLRP3, cleaved Caspase-1, IL-1β, and GSDMD-N. Data are presented as mean ±SEM (n = 3 per group). ##p < 0.01, compared to blank group; *p < 0.05, **p < 0.01, compared to LPS+ATP group

27a in the hippocampus and peripheral blood [42].
Honokiol, a bioactive polyphenolic substance, significantly shortened the immobility duration of LPS
mice in both TST and FST experiments, which wasstrongly related to decreased NF-κB activity in the
hippocampus [43]. Our work discovered that themiRNA-27a expression was reduced in the serum ofdepressed patient and rodent animals. Meanwhile,
mice experienced LPS or CSDS modeling displayed
reduced levels of miRNA-27a, SYK, and NF-κB.
These results support the critical role of themiRNA-27a/SYK/NF-κB axis in the development ofdepression. In addition, treatment with isoliquiritin
remarkably improved the depressive behaviors in
LPS and CSDS mice and attenuated NLRP3mediated pyroptosis and inflammation response. In

vitro study showed that similar to miRNA-27a
mimics, isoliquiritin enhanced miRNA-27a expression and declined SYK level in LPS and ATPstimulated primary microglia, and downregulated
protein expressions of p-NF-κB, NLRP3, cleaved
Caspase-1, IL-1β, and GSDMD-N. These findings
confirmed the regulation role of miRNA-27a/SYK/
NF-κB axis in the NLRP3 cascade activation andisoliquiritin-induced antidepressant activities. Furthermore, after treatment with miRNA-27a inhibitor,
isoliquiritin-induced therapeutic efficacy was reversed
in CSDS mice and in vitro, reflecting the requirement of miRNA-27a activation in isoliquiritin-related
antidepressant activity, and further highlighting thecritical role of miRNA-27a in the treatment ofdepression.

Li et al. Journal of Neuroinflammation(2021) 18:1

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Fig. 17 A hypothetical scheme of the molecular mechanisms underlying isoliquiritin-related antidepressant efficacy

Conclusions
In summary, our findings indicated that isoliquiritin
could significantly mitigate depressive symptoms in
mice, which was dependent on miRNA-27a/SYK/NF-κB
axis-regulated suppression of pyroptosis via NLRP3 cascade. The present study revealed the antidepressant
property isoliquiritin and its underlying mechanism andprovided novel therapeutic strategies for the treatment
of depression.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12974-020-02040-8.
Additional file 1: Figure S1. miRNA-27a mRNA expression in different
genders of depressed patients. Data are presented as mean ± SEM (n=12
per group). Figure S2. Influence of Isoliquiritin and Fluoxetine on naïve
animals. Male C57BL6/J mice were administrated with Isoliquiritin (30mg/kg) or Fluoxetine (20mg/kg) for 14 days, then behavioral tests and ELISA
assay were carried out. (a) Tail suspension test, (b) Forced swimming test,(c) Sucrose preference test, (d) TNF-α, (e) IL-1β, (f) IL-6. Data are presented
as mean ± SEM (n=10 per group).

Abbreviations
LPS: Lipopolysaccharide; ATP: Adenosine triphosphate; CSDS: Chronic social
defeat stress; GSDMD: Gasdermin D; SSRIs: Selective serotonin reuptake
inhibitors; NLRs: NOD-like receptors; CSF: Cerebral spinal fluid;
miRNA: MicroRNA; SYK: Spleen tyrosine kinase; TST: Tail suspension test;
FST: Forced swimming test; SPT: Sucrose preference test; OFT: Open field
test; SIT: Social interaction test; qRT-PCR: Quantitative real-time polymerase
chain reaction; WT: Wild-type; MUT: Mutant; PVDF: Polyvinylidene difluoride;
ELISA: Enzyme-linked immunosorbent assay; ANOVA: Analysis of varianceAcknowledgements
Not applicable
Authors’ contributions
Yuanjie Li and Hanqing Wang designed the study. Yuanjie Li, Wen Song, and
Yue Tong performed the experiments. Xia Zhang and Jianjun Zhao analyzed
the data. Yuanjie Li and Xiaojuan Gao drew the manuscript. Jingjiao Yongand Hanqing Wang revised the paper. The authors read and approved thefinal manuscript.
Funding
The study was supported by Ningxia hui autonomous region key researchand development plan general project (2018BEG03015), the National Natural
Science Foundation of China (81603227, 81460645), Key research anddevelopment projects of east-west cooperation (2017BY079), and the Third
batch of Ning xia youth talents supporting program (TJGC2018016).

Li et al. Journal of Neuroinflammation(2021) 18:1

Availability of data and materials
All data generated or analyzed during this study are included in thispublished article.
Ethics approval and consent to participate
All studies were conducted in accordance with the Provision and General
Recommendation of Chinese Experimental Animals Administration
Legislation and were approved by the ethics committee of Ningxia Medical
University.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
College of Pharmacy, Ningxia Medical University, 1160 Shengli Street,
Yinchuan 750004, Ningxia, People’s Republic of China. 2Ningxia Engineeringand Technology Research Center for Modernization of Regional
Characteristic Traditional Chinese Medicine, Ningxia Medical University,
Yinchuan, People’s Republic of China. 3Key Laboratory of Hui Ethnic Medicine
Modernisation, Ministry of Education, Ningxia Medical University, Yinchuan,
People’s Republic of China.
Received: 6 July 2020 Accepted: 17 November 2020

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