(2022) 19:41
Dang et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-022-02400-6Open Access

RESEARCH

Edaravone ameliorates depressive
and anxiety‑like behaviors via Sirt1/Nrf2/HO‑1/
Gpx4 pathway
Ruozhi Dang1,2†, Mingyang Wang1†, Xinhui Li1,2†, Haiyang Wang1,3, Lanxiang Liu1,4, Qingyuan Wu5,
Jianting Zhao6, Ping Ji7,8, Lianmei Zhong9, Julio Licinio10 and Peng Xie1,2*Abstract
Background: The inflammation and oxidative stress (OS) have been considered crucial components of the pathogenesis of depression. Edaravone (EDA), a free radical scavenger, processes strong biological activities including antioxidant, anti-inflammatory and neuroprotective properties. However, its role and potential molecular mechanisms in
depression remain unclear. The present study aimed to investigate the antidepressant activity of EDA and its underlying mechanisms.
Methods: A chronic social defeat stress (CSDS) depression model was performed to explore whether EDA could
produce antidepressant effects. Behaviors tests were carried out to examine depressive, anxiety-like and cognitive
behaviors including social interaction (SI) test, sucrose preference test (SPT), open field test (OFT), elevated plus maze(EPM), novel object recognition (NOR), tail suspension test (TST) and forced swim test (FST). Hippocampal and medial
prefrontal cortex (mPFC) tissues were collected for Nissl staining, immunofluorescence, targeted energy metabolomics analysis, enzyme-linked immunosorbent assay (ELISA), measurement of MDA, SOD, GSH, GSH-PX, T-AOC andtransmission electron microscopy (TEM). Western blotting (WB) and quantitative real-time polymerase chain reaction(qRT-PCR) detected the Sirt1/Nrf2/HO-1/Gpx4 signaling pathway. EX527, a Sirt1 inhibitor and ML385, a Nrf2 inhibitor
were injected intraperitoneally 30 min before EDA injection daily. Knockdown experiments were performed to determine the effects of Gpx4 on CSDS mice with EDA treatment by an adeno-associated virus (AAV) vector containing
miRNAi (Gpx4)–EGFP infusion.
Results: The administrated of EDA dramatically ameliorated CSDS-induced depressive and anxiety-like behaviors. In
addition, EDA notably attenuated neuronal loss, microglial activation, astrocyte dysfunction, oxidative stress damage,energy metabolism and pro-inflammatory cytokines activation in the hippocampus (Hip) and mPFC of CSDS-induced
mice. Further examination indicated that the application of EDA after the CSDS model significantly increased the protein expressions of Sirt1, Nrf2, HO-1 and Gpx4 in the Hip. EX527 abolished the antidepressant effect of EDA as well as
the protein levels of Nrf2, HO-1 and Gpx4. Similarly, ML385 reversed the antidepressant and anxiolytic effects of EDA*Correspondence: xiepeng@cqmu.edu.cn†
Ruozhi Dang, Mingyang Wang and Xinhui Li contributed equally to thiswork
1
NHC Key Laboratory of Diagnosis and Treatment on Brain Functional
Diseases, The First Affiliated Hospital of Chongqing Medical University, 1
Youyi Road, Yuzhong District, Chongqing 400016, China
Full list of author information is available at the end of the article© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Dang et al. Journal of Neuroinflammation(2022) 19:41

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via decreased expressions of HO-1 and Gpx4. In addition, Gpx4 knockdown in CSDS mice abolished EDA-generated
efficacy on depressive and anxiety-like behaviors.
Conclusion: These findings suggest that EDA possesses potent antidepressant and anxiolytic properties throughSirt1/Nrf2/HO-1/Gpx4 axis and Gpx4-mediated ferroptosis may play a key role in this effect.
Keywords: Edaravone, Depression, Anxiety, Oxidative stress, Gpx4, Ferroptosis

Background
Major depression disorder (MDD), a common, debilitating psychiatric disorder, affects approximately 17% of the
population at some point in life [1, 2]. Although there
are available antidepressants, most of the pharmacologic
interventions take weeks to months to produce effects
and have low response rates [3, 4]. Furthermore, somestriking studies have found that ketamine can produce
rapid antidepressant effects, and its s-isomer, esketamine
has been approved by the Food and Drug Administration(FDA) to treat adults with treatment-resistant depression [5, 6]. Although groundbreaking, some serious side
effects, dissociative, psychotomimetic action and abusepotential limit its wide application. Therefore, novel therapies with fewer adverse effects and higher efficacy areurgently needed.
Accumulating evidence demonstrated a close link
between oxidative stress (OS) and MDD [7, 8]. In general, OS is defined as an imbalance between the oxidation and antioxidant capacity, involving reactive oxygen
species (ROS) and reactive nitrogen species (RNS) [9].
ROS is involved in glutamate-dependent long-term
potentiation (LTP) and moderate ROS is necessary forthe growth and development of neurons [8]. Abundant studies showed that MDD was accompanied bydecreased antioxidant and increased ROS status [8, 10,11]. Moreover, preclinical and clinical evidence on OS
and antioxidant effects of antidepressants addressedthat they can scavenge ROS and RNS by scavenging free
radicals and inhibiting OS pathways [11]. This process
will protect neurons from the effect of OS and alleviate
depression. In addition, preclinical and clinical studies have also shown that inflammation plays a crucialrole in depression [12–14]. Increased pro-inflammatory
cytokines, including IL-1β, IL-6 and TNF-α were foundin MDD patients, postmortem brains with MDD androdent studies [15–17]. Silent information regulator
2 homolog 1 (Sirt1), a ­NAD+ dependent protein deacetylase, regulates acetylation of specific transcription
factors, proteins and is involved in abundant functions
including energy metabolism, stress responses, inflammation and redox homeostasis [18, 19]. Specifically,
recent studies have shown that Sirt1 plays a crucialrole in depression and regulates downstream transcription factor including nuclear factor erythroid 2-related

factor 2 (Nrf2) and NF-κB to prevent OS and inflammation damage [20–22]. Nrf2 is a primary transcription
factor in the regulation of antioxidant response and has
emerged as a potential therapeutic target for inflammatory disorders [23]. Dysregulation of Nrf2 leads tothe decrease of antioxidants and detoxifying enzymes,
which is involved in the pathogenesis of depression[24]. Heme oxygenase-1 (HO-1), a critical downstream
target stress inducible protein of Nrf2, also exerts antioxidant stress and anti-inflammatory effects [25].
Ferroptosis, a recently discovered non-caspasedependent form of programmed cell death, is characterized by the production of iron-dependent lipid ROS
and associated with OS and inflammation [26, 27]. It
differs from other types of cell death, such as apoptosis,
autophagy, necrosis, and pyroptosis in morphological,
physiological and genetically characteristics [28]. BothNrf2 and HO-1 could be inducible and participate in thesynthesis of glutathione peroxidase 4 (Gpx4), which isthe first discovered central inhibitor of ferroptosis [29].
Gpx4, a lipid repair enzyme, plays a key role in the regulation of ferroptosis. Inhibition of ­Xc− leads to depletionof cysteine and impaired the function of Gpx4, which
eventually leads to ferroptosis [30]. Other studies haveshown that Gpx4 can be up-regulated by pro-inflammatory cytokines and overexpression of Gpx4 can prevent
cell death from oxidative damage [31–33]. To the best of
our knowledge, there is still no systematic research on
the relation between ferroptosis and depression.
Edaravone(3-methyl-1-phenyl-2-pyrazolin-5-one,
EDA), a free radical scavenger, processes strong biological activities including antioxidant, neuroprotective andanti-inflammatory effects [34]. It is commonly used totreat acute ischemic stroke and acute cerebral infarction. Moreover, EDA is effective in altering amyotrophic
lateral sclerosis (ALS) progression, which is the second
approved drug for treatment of ALS [35]. Recent studies have found that EDA exerts antioxidant and antiinflammatory effects by Nrf2/HO-1 signaling pathway
in asthma and cerebral infarction [36]. Interestingly,
only a few studies have addressed EDA can attenuate
depressive-like behavior [37, 38]. However, the detailed
molecular mechanisms remain unclear. Accordingly,
we hypothesized that EDA might ameliorate depressive

Dang et al. Journal of Neuroinflammation(2022) 19:41

and anxiety-like behaviors via Sirt1/Nrf2/HO-1/Gpx4
pathway.
To investigate this hypothesis, we establishedchronic stress defeated stress (CSDS) depression micemodel and found EDA ameliorated depressive, anxiety-like behaviors and neuronal loss, affected energy
metabolism, inhibited microglial activation, OS damage and pro-inflammatory cytokines (IL-1β, IL-6 andTNF-α) activation, attenuated astrocyte dysfunction
and suppressed ferroptosis and inflammation responseby regulating the Sirt1/Nrf2/HO-1/Gpx4 pathway. Furthermore, the pivotal role of Gpx4-mediated ferroptosis
in EDA-related antidepressant and anxiolytic effects was
confirmed using Gpx4 knockdown virus.

Materials and methods
Animals and drugs treatment

Male C57BL/6J mice (aged 7–8 weeks) and retired maleCD-1 mice (aged 16–20 weeks) were obtained from theExperimental Animal Centre of Chongqing Medical University (Chongqing, China). The experimental animals
were housed in cages under a 12 h light/12 h dark cycle(lights on at 8:00 a.m.), 60 ± 5% humidity, and a temperature of 23 ± 1 °C with access to water and food freely.
All experimental procedures were conducted in accordance with the Ethics Committee of Chongqing Medical
University. EDA was purchased from Sigma-Aldrich (St.
Louis, USA) and was dissolved in Vehicle (NaCl, 0.9%)
at a dosage of 10 mg/kg. EX527 (a Sirt1 inhibitor) andML385 (a Nrf2 inhibitor) were obtained from MedChemExpress (New Jersey, USA). The doses of these threedrugs were determined according to previous studies[37–40].
The study was conducted into four experiments.
In the first experiment, mice were randomly divided
into three groups (10 each group): Control (CON) + Vehicle, CSDS + Vehicle and CSDS + EDA [10 mg/kg, intraperitoneally (i.p.)]. The experiment schedule is shown in
Fig. 1A. After 10 day CSDS model, mice were administered intraperitoneally (i.p.) daily injections of EDA or
Vehicle for 10 days followed by behavioral tests and tissue
collection.
In the second experiment, mice were randomly divided
into the following groups (n = 8 per group): CON, CSDS,
CSDS + EDA, CSDS + EDA + EX527 [10 mg/kg/d, intraperitoneally (i.p.)]. The experiment schedule is shownin Fig. 12A. EX527 was administered in CSDS-induced
mice 30 min before the administration of EDA.
In the third experiment, mice were randomly divided
into the following groups (n = 8 per group): CON, CSDS,
CSDS + EDA, CSDS + EDA + ML385 [30 mg/kg/d, intraperitoneally (i.p.)]. The experiment schedule is shown

Page 3 of 29

in Fig. 14A. ML385 was administered in CSDS exposed
mice 30 min before the administration of EDA.
To explore the Gpx4 role in CSDS-induced depression and anxiety, animals were randomized divided into
six groups (10 each group): CON, CSDS, CSDS + Vehicle, CSDS + EDA, CSDS + EDA + CON-miRNAi and
CSDS + EDA + Gpx4-miRNAi. The experiment scheduleis shown in Fig. 16A.
Chronic social defeat stress (CSDS)The CSDS paradigm was conducted as described previously [41]. Retired male CD-1 mice were screened for
3 consecutive days and the aggressors were selectedaccording to the following criteria: (i) CD1 mice attacked
the C57BL/6J mice for 2 consecutive days and (ii) the
latency to the initial attack on C57BL/6J mice was under
60 s (s). For 10 consecutive days, mice were placed in thehome cage of CD1 and were defeated for 5 min. Afterphysical defeat, a perforated divider separated them for
24 h until the next physical defeat. Each day, intruder
mice always faced a different resident. After 10 days of
CSDS, retired male CD-1 mice and experimental mice
were singly housed and performed 24 h later for thesocial interaction (SI) test.
SI test was composed of two stages. In the first stage,
the experimental mice were allowed to explore an open
field arena (44 cm × 44 cm × 30 cm) with an empty plastic box (10 cm × 7 cm × 18 cm) for 2.5 min. In the second stage, a novel, aggressive CD-1 mouse was placed in
the plastic box and was recorded for 2.5 min. The timeof mice spent in the interaction zone (IZ) surrounding
the plastic box was recorded and analyzed (Ethovision,
Noldus, The Netherlands). For SI test, an SI ratio was
calculated. SI ratio = time spent in interaction zone witha CD1 mouse/time spent in interaction zone withouta CD1 mouse. Susceptible mice were defined by SI < 1,
whereas resilient mice were defined by SI > 1. All miceused in the first experiment were susceptible mice.
Sucrose preference test (SPT)

Before the experiment, mice were habituated with two
identical water bottles for 3 days. Each mouse was waterand food-deprived for 24 h and then provided with 1%
water and sucrose solution. After 12 h, the bottles were
weighted and the sucrose preference was calculated as[sucrose water intake/(sucrose water intake + pure water
intake)] × 100%.

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 1 Effects of EDA on CSDS-induced depressive and anxiety-like behaviors. A Schematic representation of the CSDS procedure and treatments inmice. B Heat maps of path tracing in the social interaction test after EDA administration. C Social interaction test. D Sucrose preference test. E Open
field test. F Tail suspension test. G Forced swimming test. H, I Novel object recognition test. J Elevated plus maze test. All the data are expressed asmean ± SEM (n = 10 per group). **p < 0.01, ***p < 0.001 versus the CON + Vehicle group. #p < 0.05, ##p < 0.01 versus the CSDS + Vehicle group

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Open field test (OFT)

Nissl staining

A plain, 44 cm × 44 cm × 30 cm open field arena wasused to assess the locomotor activity and anxiety-likebehavior [42]. After a 30 s habituation, the total distancetraveled, time spent in center arena (14.7 cm × 14.7 cm)
was recorded in a 5 min session.

10-μm coronal cryosections were stained with Nissl
staining solution (Beyotime, C0117, Shanghai, China)
for 5 min at 37 °C. Then samples were washed using 95%
ethyl alcohol for 5 min and dried. Sections were thenwashed twice in xylene for 5 min. After being sealedwith neutral balsam, the slides were observed under an
optical microscope (Axio Image A2; Carl Zeiss, Germany) by a blinded investigator.

Elevated plus maze (EPM)

The EPM test was used to measure anxiety-related
behavior in rodents [43]. The apparatus was composedof two closed arms (30 cm × 6 cm × 15 cm) and two
open arms (30 cm × 6 cm). Animals were placed in the
EPM to explore for 5 min and time spent in closed andopen arms was calculated.
Tail suspension test (TST)

Mice were suspended by the tails by using adhesive
tape 1 cm from the tip of the tail and 15 cm above
the ground. Small plastic tubes were attached to tails
to make sure mice could not climb. The tests lasted for
6 min and the immobility time was measured and analyzed during the final 4 min.
Forced swim test (FST)

Mice were placed individually into a transparent glass
cylinder (diameter 15 cm, height 30 cm) filled with
15 cm depth of water (23 ± 1 °C). The animals wereplaced in the cylinders for 6 min and the immobility
time was calculated during the last 4 min.
Novel object recognition (NOR)

The NOR test was performed according to previous protocols [44]. Briefly, the test was performed in an open field
arena (44 cm × 44 cm × 30 cm). Objects were fixed to theopen field arena and had different shapes, sizes and textures. The NOR test consists of two stages. During a 5 minacquisition phase, the animals were placed at the center of
the arena in the presence of two identical objects (height:
12 cm; base diameter: 7 cm). After 2 h, a 5 min retrieval
phage was conducted and one of the two familiar objects
was replaced by a novel object (6.5 cm × 6.5 cm × 6.5 cm).
The time spent exploring familiar and novel objects wasrecorded and analyzed. The apparatus was cleaned with
75% ethanol after each test. The “discrimination index”
was calculated by the following [(novel object time)/(novel
object time + familiar object time)]. Object exploration
was defined when the mice touch the object with its nose
within 2 cm or less, and actively explore the objects.

Immunofluorescence and image analysis

Immunofluorescence staining was performed on frozen coronal sections of mice brains. After post-fixationand concentration dehydration, the brains were cut into
20 μm thick sections. Then the slices were washed in PBS
for 5 min three times, blocked with 5% bovine serum
albumin (BSA) and 0.2% Triton X-100 for 2 h at room
temperature. After blocking, the sections were incubated with primary antibodies overnight at 4 °C, and thensequentially incubated with the secondary antibodies for
2 h at room temperature. Primary antibodies include:
Rabbit anti-Iba-1 (1:500, Wako, 019-19741), Goat antiGFAP (1:500, Abcam, ab53554), Rabbit anti-TREM2(1:150, Proteintech, 13483-1-AP), Rabbit anti-Connexin
30 (1:200, Abcam, ab200866), Rabbit anti-Connexin 43(1:100, Thermo, 710700). The nuclei were counterstainedwith DAPI for 10 min. Fluorescent images were captured with a fluorescence microscope (VS200; Olympus,
Japan) and a confocal microscope (A1R, Nikon, Japan).
The number of cells was counted manually containing
the intact hippocampus (Hip) and medial prefrontal cortex (mPFC) from 3 mice in each group. Branch lengthand branch number of Iba-1 and GFAP positive cells
were measured using Image-Pro Plus 6.0 (Media Cybernetics, USA) and five micrographs for each mouse wereanalyzed. Areas of CX43 and CX30 staining were analyzed using Image J software.
Transmission electron microscopy (TEM)

Mice were anesthetized and perfused with phosphatebuffered solution (PBS) followed by 4% paraformaldehyde. Small Hip and mPFC tissues (1 ­mm3) were quickly
dissected and post-fixed overnight at 4 °C using 2.5%
glutaraldehyde. Tissues were embedded and cut intoalong the coronal plane at a thickness of 60–80 nm. The
mitochondrial morphology was detected by transmission electron microscope (JEM-1400 PLUS, Japan) by ablinded pathologists.
Measurement of MDA, SOD, GSH, GSH‑PX and T‑AOC

The activities of the antioxidant enzymes of malondialdehyde (MDA), the superoxide dismutase (SOD), reduced

Dang et al. Journal of Neuroinflammation(2022) 19:41

glutathione (GSH), the glutathione peroxidase (GSH-PX)
and total antioxidant capacity (T-AOC) within Hip andmPFC tissues were measured with MDA activity assay
kit (No. A003-1-2), SOD activity assay kit (No. A0013-2) and GSH activity assay kit (No. A006-2-1), GSHPX activity assay kit (No. A005-1-2) and T-AOC assay
kit (S0121, Beyotime, China). MDA, SOD, GSH and
GSH-PX kits were purchased from Jiancheng Inc. (Nanjing, China). In addition, it was necessary to pre-treat
the brain tissues before measuring the above assay kits.
Briefly, the tissues were weighted and PBS (pH 7.2–7.4,
0.01 mol/L) was added at a 1:9 proportion (MDA, SOD,
GSH and GSH-PX kits) and a 1:5 proportion for T-AOC.
Next, the samples were treated with tissue lyser (Jingxin,
China), followed by centrifuging the homogenate at
12,000 rpm for 10 min at 4 °C, and collecting the supernatant to measure.
LC–MS/MS analysis

LC–MS/MS was conducted according to previous studies [45]. Briefly, 30 mg brain tissues were preconditioned
with 200 μl cool ultrapure water for homogenization.
Then 800 μl methanol/acetonitrile (1:1, v/v) were added
and the mixture was sonicated twice for 30 min andplaced 1 h at − 20 °C to precipitate proteins. After centrifugation, the supernatants were dried and collected.
The analysis of supernatants was conducted with Agilent
1290 Infinity LC chromatography system and AB SCIEX
5500 QTRAP mass spectrometer.
Western blotting (WB)

The Hip and mPFC tissues were dissociated by radioimmunoprecipitation assay solution containing proteaseinhibitors and phosphatase inhibitors. The proteins inthe samples were run on 10% SDS polyacrylamide gel
and then transferred to polyvinylidene fluoride membrane (Millipore, USA). The proteins were incubatedwith Rabbit anti-Sirt1 (1:1000, Abcam, ab189494), Rabbit anti-Nrf2 (1:1000, Proteintech, 16396-1-AP), Rabbit
anti-HO-1 (1:1000, Proteintech, 10701-1-AP), Rabbit
anti-Gpx4 (1:3000, Abcam, ab125066), Rabbit anti-TLR4(1:1000, Abcam, ab217274), Rabbit anti-NF-κB p65(phospho S536) (1:1000, Abcam, ab76302), Rabbit antiNF-κB p65 (1:1000, Cell Signaling, 8242) and Mouse antiGAPDH (1:10,000, Abcam, ab8245) overnight at 4 °C,
and secondary anti-mouse HRP-conjugated (1:10,000,
Bio-rad, 170-6516) or secondary anti-rabbit HRP-conjugated antibodies (1:10,000, Bio-rad, 170-6515) wereincubated for 2 h at room temperature. The signals werevisualized using ECL chemiluminescence reagent (Beyotime; US Everbright Inc.).

Page 6 of 29

Table 1 Primer sequences used in the qRT-PCR
Gene

Primer sequences

Sirt1

Forward: 5′-GCT​GAC​GAC​T TC​GAC​GAC​G-3′
Reverse: 5′-TCG​GTC​AAC​AGG​AGG​T TG​TCT-3′

Nrf2

Forward: 5′-TCT​TGG​AGT​AAG​TCG​AGA​AGTGT-3′
Reverse: 5′-GTT​GAA​ACT​GAG​CGA​AAA​AGGC-3′

HO-1

Forward: 5′-AAG​CCG​AGA​ATG​C TG​AGT​TCA-3′
Reverse: 5′-GCC​GTG​TAG​ATA​TGG​TAC​AAGGA-3′

Gpx4

Forward: 5′-CTG​C TC​T TC​CAG​AGG​TCC​TG-3′
Reverse: 5′-GAG​GTG​TCC​ACC​AGA​GAA​GC-3′

β-Actin

Forward: 5′-ACT​GCC​GCA​TCC​TCT​TCC​T-3′
Reverse: 5′-TCA​ACG​TCA​CAC​T TC​ATG​ATGGA-3′

Quantitative real‑time polymerase chain reaction(qRT‑PCR)

Total RNA in Hip and mPFC lysates of mice was
extracted with TRIzol reagent (Invitrogen, Carlsbad,
USA) and reverse transcribed into cDNA by PrimeScript
RT reagent Kit (Takara, Japan) following the manufacturer’s guides. Relative mRNA expression levels were
performed with SYBR Green detection system (Roche,
Germany). All samples were performed in triplicate
and normalized with β-actin mRNA level. The primer
sequences are listed in Table 1.
Enzyme‑linked immunosorbent assay (ELISA)

The levels of TREM2, GFAP, IL-1β, IL-6 and TNF-α inthe Hip and mPFC were measured by ELISA kits (Jianglai
Biological, Shanghai, China). The tissues were weighed,and PBS was added at a 1:9 proportion and homogenized. Then, the homogenate was centrifuged at5000 rpm for 15 min, and collecting the supernatant tomeasure. According to the instructions, 100 μl standard/sample was added and incubated for 1 h at 37 °C. Then,biotin antibody was added into each well and incubatedfor 1 h at 37 °C. The plate was then washed with washing buffer and streptavidin–HRP was added for 30 min at
37 °C. Next, TMB substrate was added to each well andkept away from light for 15 min at 37 °C after washing.
Finally, stop solution was added and the optical density
was measured at 450 nm.
Stereotaxic virus injection

Mice were anesthetized with isoflurane and thenplaced on a stereotaxic apparatus (RWD, Shenzhen,
China). Virus was injected using Nanoject II (Drummond Scientific, PA) via a micro pipette. 3 min after
pipette insertion, small amounts of virus were multiple injected at 30 s intervals (13 nl/s). After waiting for 10 min, the pipette was slowly retracted. 2 μl
AAV2/8-CMV-EGFP-miRNAi(Gpx4) or AAV2/8-CMVEGFP-miRNAi(NC) (Taitool, Shanghai, China) wasDang et al. Journal of Neuroinflammation(2022) 19:41

bilaterally injected into the Hip (coordinates, bregma:
AP = − 1.70 mm; ML =  ± 1.25 mm; DV = − 1.20 mm,
bregma: AP = − 3.15 mm; ML =  ± 2.94 mm; DV = − 3.62,− 2.25 mm). Behavioral tests were commenced 4 weeks
after viral injection.
Statistical analysis

Data were presented as mean ± SEM and analyzed with
GraphPad Prism 8.0 or the statistical program SPSS 17.0.
The normality of data was assessed individually and nonparametric test were used when p < 0.05. Homogeneity ofvariance was be verified by Brown–Forsythe test, whenp < 0.05, data were analyzed by one way analysis of variance (ANOVA) followed by Dunnett’s T3 post hoc test.
Statistical comparisons among groups were performedby using ANOVA followed by Tukey’s test or Fisher’s LSD(data for energy metabolites in Fig. 9) post hoc test fornormally distributed data and Kruskal–Wallis test withmultiple comparisons for non-normally distributed data.
p < 0.05 was regarded as significant.

Results
EDA ameliorates depression and anxiety‑related behaviors
after CSDS exposure

To investigate the effect of EDA on depression, we
exposed mice to a well-established animal model ofdepression, CSDS, and subsequently performed multiple
behavioral tests including SI, SPT, OFT, NOR, EPM, TST,and FST (Fig. 1A). As shown in Fig. 1B, C, mice exposed
to CSDS showed significantly decreased SI ratio. However, intraperitoneal injection of EDA improved the SI
ratio as compared to the CSDS + Vehicle group. In SPT,the consumption of sucrose was significantly reduced
in CSDS exposed mice compared with the control mice.
Nevertheless, EDA intervention greatly reversed CSDSinduced reduction in sucrose preference, which reflectedanhedonia (Fig. 1D). Next, mice, exposed to an inescapable aversive environment TST and FST, initially violent
escape but then transition to a passive coping (PC) state[46]. This state can be intervened by pharmacologicalapproach and may be associated with the pathologicalmotivational impairments in MDD [47]. We found that
CSDS + Vehicle animals exhibited robust increase immobility in TST and FST. As expected, CSDS + EDA mice
exhibited significant decreases in time spent immobile
compared to CSDS + Vehicle mice (Fig. 1F, G). These
results consistently indicated that EDA elicits antidepressant effect in mice.
In addition, we employed OFT and EPM to evaluate
anxiety-like behavior. The CSDS + Vehicle mice exhibited remarkable anxiety-like behavior relative to the controls, as indicated by less time spent in the center zone ofPage 7 of 29

OFT and the open arms in the EPM. Nevertheless, thisreduction was markedly reversed by the administrationof EDA, indicating EDA was effective in ameliorating
CSDS-evoked anxiety (Fig. 1E, J).
Previous study has observed oral administrationof EDA ameliorated the Alzheimer’s disease (AD)like pathologies and memory deficits of the mice [48].
Accordingly, we carried out the NOR test to determine
whether EDA affects the recognition memory of mice
(Fig. 1H, I). Interestingly, there was no difference in timespent with the novel object among all groups, displayed
no impairment of object memory retention.
EDA treatment significantly alleviates neuronal injury

MDD was thought to be primarily triggered through neuronal dysfunction in the Hip and mPFC [49]. Based on
existing evidence, Nissl staining was employed to observe
neuronal morphological changes in the Hip and mPFC.
As shown in Fig. 2A, C, neurons in the hippocampal CA1
and mPFC regions were arrayed regularly and compactly
and Nissl bodies were clear in the CON + Vehicle group.
Whereas, CSDS-evoked mice have increased damaged
neurons, which were irregular and sparsely distributed
as well as Nissl body disintegrated compared with the
CON + Vehicle group. The number of Nissl-positive cells
was observably reduced compared with the CON + Vehicle group. After EDA administration, the decrease of
Nissl-positive neurons and neuronal damage were ameliorated (Fig. 2B, D). These data indicated that EDA could
attenuate CSDS-induced neuronal damage.
Administration of EDA inhibits microglial activation
and attenuates astrocyte dysfunction in the CSDS exposed
mice

Neuroinflammation is tightly associated with the onset
of depression [50, 51]. Given that microglia and astrocytes play an essential role in neuroinflammation andundergo abnormally structural and functional changes
in the Hip and mPFC in response to chronic stress, wefirst examined the effect of EDA on the distribution
and morphology of microglia in the Hip and mPFC
using ionized calcium-binding adaptor molecule 1 (Iba1) staining (Fig. 3A, E). The hippocampal and mPFC
microglia exhibited striking activation morphology after
CSDS exposure, as indicated by increased cell number,
total branch length and branch number. These effects
were significantly alleviated by EDA administration(Fig. 3B–D, F–H). The triggering receptor expressed on
myeloid cells 2 (TREM2) is an innate immune receptor
and uniquely expressed by microglia in the brain [52].
Mounting evidence have indicated TREM2 regulates
inflammatory responses mediated by microglia [53, 54].

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 2 Influence of EDA on neuronal death in the Hip and mPFC. A, B Nissl staining in the hippocampal CA1 subregion and its statistical analysis. C,
D Nissl staining in the mPFC and its statistical analysis. Data are presented as mean ± SEM (n = 3 per group). Scale bar, 50 μm. **p < 0.01, ***p < 0.001
versus the CON + Vehicle group. #p < 0.05 versus the CSDS + Vehicle group

To convincingly verify the key role of microglial activation in CSDS-induced mice, the TREM2 was detected by
Immunofluorescence and ELISA. As shown in Fig. 4A,
B, we found that the expression of TREM2 was dramatically reduced in the CSDS + Vehicle mice compared withthat of CON + Vehicle mice in the Hip. After treatment
with EDA, the TREM2 expression level was markedly upregulated in the CSDS mice. Similar phenotype was also
identified in the mPFC region (Fig. 4C, D). In addition,
decreased protein level of TREM2 induced by CSDS was
remarkably increased by EDA treatment in the Hip andmPFC through ELISA kit (Fig. 4E, G).
Since CSDS mice experience neuroinflammation leading to astrocyte dysfunction, we then examined the effectof EDA on astrocyte morphology and functional change.
The histological change was confirmed by glial
fibrillary acidic protein (GFAP) staining (Fig. 5A, E).
CSDS exposure resulted in astrocyte dysfunction in
CSDS + Vehicle mice, as indicated by the fact that CSDS
exposure decreased the number of GFAP-positive cells
and by the ramification of astrocytes characterized by
the significantly decreased branch numbers and length.
As expected, administration of EDA remarkably alleviated astrocyte dysfunction (Fig. 5B–D, F–H). In addition,
ELISA results also showed that CSDS stimuli dramaticallydecreased the protein level of GFAP in the Hip and mPFC
(Fig. 4F, H). After EDA administration, the expression of
GFAP was significantly increased in the Hip. There wasan increased tendency in CSDS + EDA group as compared to CSDS + Vehicle group in the mPFC of GFAP.
Mounting evidence certificated that astrocytic network
proteins Cx43 and Cx30 are primarily expressed in astrocytes and help to regulate ion communication between
astroglial activation and astroglial secretion [55, 56]. We
observed down-regulation of astrocytic network markers Cx43 and Cx30 in CSDS exposed mice, while treatment with EDA significantly increased the expression of
Cx43 and Cx30 in the Hip and mPFC (Fig. 6).Together,
these results suggested that the microglial and astrocytic
abnormality in CSDS exposed mice can be restored by
the replenishment of EDA, suggesting a potential therapeutic strategy for depression.
Effects of EDA on OS and mitochondrial damage in the Hip
and mPFC

Given OS is emerging as an important therapeutic target for MDD, we measured the activities of MDA, SOD,
GSH, GSH-PX and T-AOC in the Hip and mPFC to evaluate the effect of EDA on OS induced lipid peroxidation.

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 3 Effects of EDA on microglial activation in the CSDS mice Hip and mPFC. A EDA inhibited microglial activation in the CSDS mice Hip.
Representative images of microglia immunostaining for Iba-1. Scale bars, 100 μm (upper panel) and 10 μm (lower panel). B Quantification of Iba-1
positive cells per m­ m2 in the mice Hip. C Total branch length. D Branch number. E EDA inhibited microglial activation in the CSDS mice mPFC.
Representative images of microglia immunostaining for Iba-1. Scale bars, 100 μm (upper panel) and 10 μm (lower panel). F Quantification of Iba-1
positive cells per m­ m2 in the mPFC. G Total branch length. H Branch number. All the data are presented as mean ± SEM (n = 3 mice/group, 15 cells/
group). *p < 0.05, ***p < 0.001 versus the CON + Vehicle group. #p < 0.05, ##p < 0.01 versus the CSDS + Vehicle group

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Fig. 4 Administration of EDA increased the expression of TREM2 and GFAP. A, B Immunofluorescence staining of TREM2 in the Hip. Scale bar,
50 μm. C, D Immunofluorescence staining of TREM2 in the mPFC. Scale bar, 50 μm. E, F Concentrations of Trem2 and GFAP in the Hip. G, H
Concentrations of TREM2 and GFAP in the mPFC. Data are presented as mean ± SEM (n = 3 per group for immunostaining experiments, n = 6 micefor ELISA). **p < 0.01, ***p < 0.001 versus the CON + Vehicle group. #p < 0.05, ##p < 0.01, ###p < 0.001 versus the CSDS + Vehicle group

As shown in Fig. 7A, B, D, E, the activities of SOD, MDA,
GSH-PX and T-AOC were significantly decreased in theHip of CSDS + Vehicle mice. In contrast, the content of
GSH in the CSDS + Vehicle group was notably higher
than the CON + Vehicle group. However, intraperitoneal injection of EDA completely reversed these alterations (Fig. 7A–E). In the mPFC tissue, CSDS exposure

significantly decreased MDA, GSH-PX and T-AOC
levels. After EDA application, mice in the CSDS + EDA
group recovered MDA, GSH, GSH-PX and T-AOC
activities (Fig. 7G–J). Nevertheless, SOD activity had nochanges among three groups (Fig. 7F).
Since the antioxidant property of EDA was mitochondria-mediated, we used TEM to observe mitochondrial damages in the Hip and mPFC. CSDS exposure
exhibited profound effects on mitochondrial structure,

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 5 Effects of EDA on astrocyte loss in the CSDS mice Hip and mPFC. A Effect of EDA on astrocyte dysfunction-induced by CSDS in the Hip.
Representative images of astrocyte immunostaining for GFAP. Scale bars, 100 μm (upper panel) and 10 μm (lower panel). B Quantification of
GFAP-positive cells per m­ m2 in the Hip. C Total branch length. D Branch number. E Effect of EDA on astrocyte loss-induced by CSDS in the mPFC.
Representative images of astrocyte immunostaining for GFAP. Scale bars, 100 μm (upper panel) and 10 μm (lower panel). F Quantification of
GFAP-positive cells per m­ m2 in the Hip. G Total branch length. H Branch number. All the data are presented as mean ± SEM (n = 3 mice/group, 15
cells/group). *p < 0.05, ***p < 0.001 versus the CON + Vehicle group. #p < 0.05, ##p < 0.01 versus the CSDS + Vehicle group

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 6 Administration of EDA increased the expression of astrocytic network proteins Cx43 and Cx30 in the Hip and mPFC. A, B
Immunofluorescence staining of GFAP and Cx43 in the Hip. Scale bar, 50 μm. C, D Immunofluorescence staining of GFAP and Cx43 in the mPFC.
Scale bar, 50 μm. E, F Immunofluorescence staining of GFAP and Cx30 in the Hip. Scale bar, 50 μm. G, H Immunofluorescence staining of GFAP andCx30 in the mPFC. Scale bar, 50 μm. Data are presented as mean ± SEM (n = 3 per group). **p < 0.01, ***p < 0.001 versus the CON + Vehicle group.
#p < 0.05, ##p < 0.01 versus the CSDS + Vehicle group

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Fig. 7 Changes of OS parameters in the Hip and mPFC after EDA exposure. A–E Levels of OS parameters including MDA, SOD,GSH,GSH-PX and
T-AOC in the Hip. F–J MDA content, the activities of SOD, GSH, GSH-PX and T-AOC in the mPFC. Data are presented as mean ± SEM (n = 6 pergroup). *p < 0.05, **p < 0.01, ***p < 0.001 versus the CON + Vehicle group. #p < 0.05, ##p < 0.01, ###p < 0.001 versus the CSDS + Vehicle group

including mitochondrial swelling and loss of mitochondrial cristae in neurons (Fig. 8) and microglia (Additional file 1: Fig. S1). In addition, compared with the
CSDS + Vehicle group, EDA treatment alleviated thesepathological changes. Interestingly, mitochondrial
damage in the Hip was more severe than in the mPFC

region (Fig. 8A, B; Additional file 1: Fig. S1a, b). Altogether, these findings implied that EDA might ameliorate CSDS-induced OS and mitochondrial damage.

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 8 Ultrastructure of neurons in the CSDS model of Hip and mPFC with EDA treatment. A Electron micrographs showed mitochondrial damages
in the hippocampal neuron (red arrows) Scale bars, 2 μm (upper panel) and 500 nm (lower panel). B Electron micrographs showed mitochondrial
damages in the mPFC neuron (red arrows). Scale bars, 2 μm (upper panel) and 500 nm (lower panel)(See figure on next page.)
Fig. 9 Targeted energy metabolomics in the Hip and mPFC of CSDS model mice. A Heatmap of partly energy-related metabolites in the Hip. B
Heatmap of partly energy-related metabolites in the mPFC. Rows indicate metabolites. The degree of change in metabolite concentration is color
coded. C–G Differential energy metabolites in the Hip. H–M Differential energy metabolites in the mPFC. Data are presented as mean ± SEM (n = 6
per group). *p < 0.05, **p < 0.01 versus the CON + Vehicle group. #p < 0.05, ###p < 0.001 versus the CSDS + Vehicle group. &p < 0.05, &&p < 0.01 versus
the CSDS + EDA group

EDA treatment affects energy metabolites in the Hip
and mPFC

Given EDA was a potent mitochondrial antioxidant andmetabolism enhancer, we carried out targeted energy
metabolomics analysis by detecting 32 metabolites in the
Hip and mPFC tissues of mice. The heat maps of metabolites changes of CON + Vehicle versus CSDS + Vehicle versus CSDS + EDA are shown in Fig. 9A, B. In theHip, the guanosine monophosphate (GMP), nicotinamide adenine dinucleotide phosphate (NADP), guanosine diphosphate (GDP) and adenosine monophosphate(AMP) in the CSDS + Vehicle group were reduced related
to the CON + Vehicle group without statistical significance. However, these metabolites were significantly
increased in the CSDS + EDA group compared with
CSDS + Vehicle group (Fig. 9C–F). In addition, Betad-Fructose 6-phosphate in the CSDS + Vehicle group wasnotably diminished compared with CON + Vehicle group(Fig. 9G). In the mPFC, we found most of the energyrelated metabolites were not changed by CSDS exposure. Nevertheless, Succinate and adenosine diphosphate(ADP) were significantly decreased in CSDS + Vehicle

Dang et al. Journal of Neuroinflammation

Fig. 9 (See legend on previous page.)(2022) 19:41

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Dang et al. Journal of Neuroinflammation(2022) 19:41

group compared with CON + Vehicle group (Fig. 9H,
I). Meanwhile, compared with CSDS + Vehicle group, 4
metabolites (GMP, ADP, AMP and GDP) were markedly
increased in the CSDS + EDA group (Fig. 9I–L). Amongthe differentially altered metabolites, GMP, GDP and
AMP were altered in both the Hip and mPFC tissues,
with the other metabolites altered either in Hip or mPFC.
Then, we used Ingenuity Pathway Analysis (IPA) to
identify the top canonical signaling pathways affected in
the Hip and mPFC with or without EDA treatment. Eachof the top 5 canonical pathways was exhibited in Additional file 2: Fig. S2. Of all these pathways, we noticed
that NAD salvage pathway II in the Hip was associatedwith the antioxidant mechanism of EDA. The NAD salvage pathway was the main source of N
­ AD+ biosynthesis
in mammalian cells [57]. Interestingly, Sirt1 is an ­NAD+
dependent histone deacetylase and mediates the levels ofanxiety and depression [58].
Furthermore, we used Kyoto Encyclopedia of Genes
and Genomes (KEGG) database to reveal the function ofdifferential metabolites in signaling pathways. Remarkably, the significant alternation of NADP in the Hip
might indicate a potential relevance between glutathione
metabolism and depression.
Taken together, these results demonstrated that EDA
administration significantly affected energy metabolites
in the Hip and mPFC. Furthermore, changes in hippocampal energy metabolites might be more associatedwith depression in contrast with mPFC.
EDA reverses Sirt1/Nrf2/HO‑1/Gpx4 pathway and prevents
CSDS‑induced neuroinflammation

Given Gpx4 plays a crucial role in glutathione metabolism and the involvement of Sirt1 and Nrf2 in MDD, wenext sought to examine the role of Sirt1/Nrf2/HO-1/
Gpx4 pathway in EDA-related depression and anxiety by
WB. As shown in Fig. 10A–G, CSDS exposure robustly
decreased the expression levels of Sirt1, Nrf2, HO-1 andGpx4 in the Hip. However, these effects were significantly
reversed by the treatment with EDA. In the mPFC, theprotein expression level of Sirt1 was significantly higher
in CSDS + EDA group as compared to the CSDS + Vehicle group (Fig. 10I). Nevertheless, the CSDS + Vehicle
and CSDS + EDA groups had no significant effects on thelevels of Nrf2, HO-1 and Gpx4 (Fig. 10J–L).
Since TLR4/NF-κB signaling pathway mediated neuroinflammation plays a key role in neuropsychiatric disorders including MDD [12]. The expressions were detected
by WB. As presented in Fig. 10A, F–H, M, N, the expression of TLR4 and p-NF-κB were dramatically increased
following CSDS stimulation, which were all reversed
by EDA administration. In addition, increased levelsof IL-1β, IL-6 and TNF-α in the Hip and mPFC tissues

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induced by CSDS were remarkably suppressed by EDA
injection (Fig. 11).
In order to evaluate Sirt1/Nrf2/HO-1/Gpx4 pathway
in transcription level, we further examined the mRNA
expressions of Sirt1, Nrf2, HO-1, and Gpx4 in the Hip
and mPFC tissues. As exhibited in Additional file 3: Fig.
S3, CSDS exposure did not change the expressions of
Sirt1 in the Hip and Gpx4 in both regions. In addition,
CSDS + Vehicle group was found with significantly upregulated mRNA (Nrf2 and HO-1) in the Hip and mPFC
as compared to CON + Vehicle group. Rather, within
exposure to EDA, we found decreased expressions ofNrf2 and HO-1 in both Hip and mPFC compared with
CSDS + Vehicle group.
Taken together, these results suggested that EDA
reverses Sirt1/Nrf2/HO-1/Gpx4 pathway and abolishes
CSDS-induced neuroinflammation.
Blockade of Sirt1 abolishes the antidepressant effectof EDA via Sirt1/Nrf2/HO‑1/Gpx4 pathway

To investigate the effects of Sirt1 on the antidepressantand anxiolytic effects of EDA, EX527 (a Sirt1 inhibitor) was administered in CSDS-induced mice 30 min
before the administration of EDA (Fig. 12A). As shownin Fig. 12B–E, H, compared with the CSDS group, SI
ratio, sucrose consumption, center time and open arms
time increased significantly after the administration of
EDA. However, when EDA and EX527 were administered together, EX527 notably reduced the EDA- induced
increase in SI ratio, sucrose consumption and discrimination index, but not in center time and open arms time.
In Fig. 12F, G, treatment with EX527 increased immobility time in TST and FST compared with those in
CSDS + EDA group.
To further validate the role of Sirt1in EDA-mediatedantidepressant effect, the proteins in the Hip, including
Sirt1, Nrf2, HO-1 and Gpx4 level were measured using
WB. As shown in Fig. 13, the expression of Sirt1, Nrf2,
HO-1 and Gpx4 was significantly higher in CSDS + EDA
mice as compared to the CSDS  group. However, inhibition of Sirt1 with EX527 abolished the effects of EDA,
which led to a decrease in Sirt1, Nrf2, HO-1 and Gpx4
levels as compared to the CSDS + EDA group.
Taken together, these results suggested that inhibitionof Sirt1 could suppress the antidepressant effect of EDA
via Sirt1/Nrf2/HO-1/Gpx4 pathway.
Blockade of Nrf2 abolishes the antidepressantand anxiolytic effects of EDA via Sirt1/Nrf2/HO‑1/Gpx4
pathway

To further evaluate whether Nrf2 is a downstream
target of Sirt1, mice were treated with Nrf2 inhibitor
ML385 (Fig. 14A). Treatment with EDA increased SI

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Fig. 10 Effect of EDA on Sirt1/Nrf2/HO-1/Gpx4 and TLR4/NF-κB signaling pathways. A Representative WB bands in the hippocampal region. B–G
Statistical graphs of relative protein expression of Sirt1 (B), Nrf2 (C), HO-1 (D), Gpx4 (E), TLR4 (F), and p-NF-κB (G). H Representative WB bands inthe mPFC region. I–N Statistical graphs of relative protein expression of Sirt1 (I), Nrf2 (J), HO-1 (K), Gpx4 (L), TLR4 (M), and p-NF-κB (N). Data arepresented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001 versus the CON + Vehicle group. #p < 0.05, ##p < 0.01, ###p < 0.001 versus
the CSDS + Vehicle groupratio, sucrose consumption, center time and open arms
time and decreased immobility time in TST and FST
compared with those in the CSDS group. On the other
hand, compared to CSDS + EDA group, the ML385
treatment markedly decreased SI ratio, sucrose consumption, center time, open arms time and discrimination index and increased immobility time in TST and

FST (Fig. 14B–H). Next, we explored Sirt1, Nrf2, HO-1
and Gpx4 expressions in the Hip after ML385 treatment. As described in Fig. 15, in response to CSDS
exposure, we observed a decrease in the protein levelsof Sirt1, Nrf2, HO-1 and Gpx4. Treatment with EDA
resulted in a significant up-regulation of those proteins.

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 11 Effect of EDA on concentrations of IL-1β, IL-6, and TNF-α in the Hip and mPFC. A–C Concentrations of IL-1β (A), IL-6 (B), and TNF-α (C) inthe hippocampal region. D–F Concentrations of IL-1β (D), IL-6 (E), and TNF-α (F) in the mPFC region. Data are presented as mean ± SEM (n = 6 pergroup). **p < 0.01, ***p < 0.001 versus the CON + Vehicle group. #p < 0.05, ##p < 0.01 versus the CSDS + Vehicle group

However, application of ML385 abolished the effects of
EDA on Nrf2, HO-1 and Gpx4, while it had no significant effect on the expression of Sirt1. These results confirmed that Nrf2 is the downstream target of Sirt1.
Blockade of Gpx4 abolishes the antidepressantand anxiolytic effects of EDA

Previous studies and our results have confirmed that
Sirt1 and Nrf2 played an important role in depression [24, 59]. It remains unknown, however, whether
Gpx4-regulated ferroptosis affect the antidepressantand anxiolytic effects of EDA. Accordingly, an adenoassociated virus (AAV) vector that selectively expresses

Gpx4–miRNAi with enhanced green fluorescent protein (AAV–EGFP–Gpx4–miRNAi) was injected into
the Hip of mice. Then CSDS depression model was
performed, followed by behavior tests by SI, SPT, OFT,NOR, EPM, TST, and FST (Fig. 16A, B). As shownin Fig. 16C–F, mice exposed to EDA showed markedly increased SI ratio in SI, sucrose consumption in
SPT, center time in OFT and open arms time in EPM
as compared to the CSDS group, whereas comparedto the CSDS + EDA group, the AAV–EGFP–Gpx4–
miRNAi treatment decreased SI ratio, center timeand open arms time, but not sucrose consumption.
As shown in Fig. 16G, H, immobility time in TST andDang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 12 Blockade of Sirt1 abolished the antidepressant and anxiolytic effects of EDA. A Schematic timeline of the experimental procedure. B
Social interaction test. C Sucrose preference test. D Open field test. E Elevated plus maze test. F Tail suspension test. G Forced swimming test. H
Novel object recognition test. All the data are expressed as mean ± SEM (n = 8 per group). **p < 0.01, ***p < 0.001 versus the CON group. #p < 0.05,##
p < 0.01, ###p < 0.001 versus the CSDS group. &p < 0.05 versus the CSDS + EDA group

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 13 Impact of Sirt1 inhibitor on EDA-induced Sirt1/Nrf2/HO-1/Gpx4 signaling pathway in the Hip of CSDS mice. A Representative WB bands.
B–E Protein levels of Sirt1 (B), Nrf2 (c), HO-1 (D), and Gpx4 (E) in the Hip. Data are presented as mean ± SEM (n = 3 per group). *p < 0.05, **p < 0.01,***p < 0.001 versus the CON group. ##p < 0.01, ###p < 0.001 versus the CSDS group. &&p < 0.01, &&&p < 0.001 versus the CSDS + EDA group

FST was significantly decreased in the CSDS + EDA
group as compared to the CSDS group. However, compared to the CSDS + EDA group, immobility time in
TST and FST in the CSDS + EDA + Gpx4–miRNAi
group was dramatically increased. Intriguingly, thediscrimination index exhibited markedly decreasedin the CSDS + EDA + Gpx4–miRNAi group as compared to the CSDS + EDA group (Fig. 16I). These
data indicated that the antidepressant and anxiolytic
effects of EDA in CSDS-induced mice were blocked by
AAV–EGFP–Gpx4–miRNAi.

Discussion
In the present study, we demonstrated that the antidepressant and anxiolytic properties of EDA and its potential mechanisms (Fig. 17). Current studies indicated that
EDA attenuated CSDS-evoked depressive symptoms

and CSDS-induced anxiety behaviors, and the underlying mechanisms were associated with the alteration of
expression of Sirt1/Nrf2/HO-1/Gpx4 pathway in theHip. The knockdown of Gpx4 in the Hip abolished theeffects of EDA treatment. Furthermore, EDA protected
microglia and astrocyte against CSDS-induced inflammation alternations, attenuated neuronal damage andpro-inflammatory cytokines activation in the Hip andmPFC. These results shed light on the antidepressant andanxiolytic effects of EDA and provide a new idea about
the Gpx4-regulated ferroptosis in depression and anxiety.
To the best of our knowledge, this study is the firstto demonstrate that the antidepressant and anxiolytic
effects of EDA were brain region-specific. EDA is a free
radical scavenger that can pass through the blood-brain
barrier (BBB) and has therapeutic effects on stroke and
ALS [35]. Only a few studies indicated that high concentrations of EDA presented antidepressant-like activity

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 14 Blockade of Nrf2 abolished the antidepressant and anxiolytic effects of EDA. A Schematic timeline of the experimental procedure. B
Social interaction test. C Sucrose preference test. D Open field test. E Elevated plus maze test. F Tail suspension test. G Forced swimming test. H
Novel object recognition test. All the data are expressed as mean ± SEM (n = 8 per group). **p < 0.01, ***p < 0.001 versus the CON group. #p < 0.05,##
p < 0.01, ###p < 0.001 versus the CSDS group. &p < 0.05 versus the CSDS + EDA group

Dang et al. Journal of Neuroinflammation(2022) 19:41

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Fig. 15 Impact of Nrf2 inhibitor on EDA-induced Sirt1/Nrf2/HO-1/Gpx4 signaling pathway in the Hip of CSDS mice. A Representative WB bands.
B–E Protein levels of Sirt1 (B), Nrf2 (C), HO-1 (D), and Gpx4 (E) in the Hip. Data are presented as mean ± SEM (n = 3 per group). *p < 0.05, **p < 0.01,***p < 0.001 versus the CON group. #p < 0.05, ###p < 0.001 versus the CSDS group. &&p < 0.01, &&&p < 0.001 versus the CSDS + EDA groupin chronic restraint stress and corticosterone model ofdepression [37, 38]. Here, we first evaluated the effectof EDA on CSDS-induced depressive behaviors. As
expected, administration of EDA ameliorated depressive
and anxiety-like behaviors.
Recent studies confirmed that MDD was associatedwith hippocampal and mPFC structural aberrations,
including cellular damage, volumetric reductions andreduced hippocampal neurogenesis [60, 61]. Thus, we
focused on Hip and mPFC regions and sought to explore
whether the antidepressant and anxiolytic effects of EDA
were present in specific region of the brain. Here, we
found that the anti-neuroinflammation effect of EDA in
CSDS-induced depressed mice was associated with inhibiting the increase of microglial activation and mitigating astrocyte dysfunction in the Hip and mPFC regions.
In addition, the neuronal death and pro-inflammatory

cytokines activation in the Hip and mPFC were relieved
with treatment of EDA. MDD was initially speculated
to be a disease derived from neuronal dysfunction [49].
However, numerous studies had linked astrocyte dysfunction to the pathophysiology of MDD [62]. In rodent
depression models or postmortem patients with MDD
patients, the expression of GFAP reduced in the Hip andprefrontal cortex as compared to controls. The reason
for reduced astrocytes in CSDS mice may be related topotassium imbalance. Chronic stress, leading to depression, can up-regulate the inwardly rectifying potassiumchannel, ­Kir 4.1 expression in astrocytic membrane processes that wrap tightly around the neuronal soma [63].
This channel on astrocytes tightly regulates the degreeof membrane hyperpolarization and burst firing of neurons, which may implicate astrocytes with depression.
Then, astrocytes are lost. This will destabilize glutamate

Dang et al. Journal of Neuroinflammation(2022) 19:41

homeostasis by reducing glutamate reuptake and synthesis. Furthermore, the decrease in astrocyte densities isalso related to decreased expression of CX30 [64].
OS and inflammation are interdependent and functionally complementary, which are ubiquitous in MDD.
Mounting evidence indicated that inflammation may
affect mitochondrial function, membrane polarity andoxidative phosphorylation, which may further lead to OS
and apoptosis [11, 65–67]. Microglia and astrocytes aremain mediators of inflammation in the brain [68]. In ourstudy reported here, the EDA administration significantly
decreased microglial activation and attenuated astrocyte dysfunction. In addition, our results were consistent with previous studies showing that the chronic stress
exposure decreased the activities of SOD, GSH-PX and
T-AOC and increased GSH activity, indicating a direct
involvement of OS in depression [69]. However, treatment with EDA alleviated this damage. Ferroptosis is oxidative damage-related regulated cell death, mainly driven
by iron accumulation, lipid peroxidation, and subsequentplasma membrane rupture [70]. Surprisingly, comparedwith previous studies that showed elevated MDA activity in depression, our results showed that MDA activity
was lower in CSDS model mice compared to controls.
Some studies also reported divergent results on MDA
level. Dionisie et al. showed that there was no significant
increase in the MDA level of frontal cortex under chronic
unpredictable mild stress (CUMS) model exposed rats[71]. Similarly, Wang et al. also demonstrated that thelevel of MDA in the Hip and cortex did not change in
the CUMS-induced rats as compared to controls [72].
Bai et al. reported that the MDA level slightly reduced in
the serum of schizophrenia patients compared with controls [73]. Decreased MDA level has also been reported
in other diseases. Ma et al. showed a decrease of serum
MDA level in gastric cancer patients compared withthose in control subjects [74]. Punnonen et al. founddecreased tissue MDA level in human breast cancer[75]. Similarly, Chang and Biasi et al. showed decreased
serum MDA level in colon cancer [76, 77]. Murugan et al.
showed significantly decreased serum MDA level in gastric carcinogenesis in rat model [78].
However, the reason for the discrepancy about MDA
is elusive. Mounting studies showed that lipid peroxidation plays a central role in mediating ferroptosis. MDA,
4-hydroxynonenal (4-HNE) and 8-hydroxydeoxyguanosine (8-OH-dG), the final products of lipid peroxidation,

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have been applied in various samples in vitro and in vivo
[79]. The MDA assay kits we used started with its measurement a component of thiobarbituric acid-reactive
substances (TBARS) for the assessment of lipid peroxidation by spectrophotometry or fluorimetry. However, thismethod lacks specificity, because compounds containing many chemically reactive carbonyl groups in different types of substances can react with thiobarbituric acid
(TBA) and interfere with MDA analysis [80]. These reactive carbonyl-containing compounds include oxidized
polyunsaturated fatty acids and carbohydrates fromendogenous sources and food in body fluids. Therefore,
the specificity and sensitivity using MDA assay kit arecontroversial, which may affect the interpretation of theresults. Therefore, using TBA as a sole indicator of lipid
peroxidation is not appropriate.
Increasing evidence indicated that mitochondrial dysfunctions have been deemed as the underlying mechanism of MDD and the increase of ROS is associatedwith the reduction in neuronal metabolism [81, 82]. This
metabolic deterioration is associated with reduced activity of ATP synthesis, primarily due to mitochondrial dysfunction. According to the “mitochondrial bioenergetics
hypothesis”, depression is the most common psychiatricdisorder in patients with impaired mitochondrial functions [82]. Given the most prominent role of mitochondria is the production of energy, we then performed TEM
and targeted energy metabolomics to further explore themitochondrial dysfunctions and abnormal energy metabolism in the Hip and mPFC tissues of depression. Previous works have shown significant disturbances of energy
metabolism in CSDS, CUMS, learned helplessness, andchronic restraint stress model of mice [45, 83]. In ourstudy, intraperitoneal injection of EDA greatly improved
the mitochondrial and energy metabolism dysfunctions.
Of note, mitochondrial damage in the Hip was more
severe than in the mPFC region.
The changes of energy metabolism pathway are
involved in the Krebs cycle and glycolysis. We thenused IPA and KEGG to analyze the differentiallyexpressed metabolites. It is worth noting that NAD salvage pathway II through IPA and glutathione metabolism
via KEGG pathway enrichment in the Hip might be associated with the antioxidant mechanism of EDA. Sirt1, a­NAD+-dependent deacetylase, plays a key role in NAD
salvage pathway II and mediates the levels of anxiety
and depression [58, 59]. Sirt1 expression was markedly(See figure on next page.)
Fig. 16 Knockdown Gpx4 in the Hip antagonized the antidepressant and anxiolytic effects of EDA. A Schematic timeline of the experimental
procedure. B Fluorescence image of a fixed brain section that expressed AAV2/8-CMV–EGFP–miRNAi(Gpx4) in the Hip. Scale bar, 500 μm. C Social
interaction test. D Sucrose preference test. E Open field test. F Elevated plus maze test. G Tail suspension test. H Forced swimming test. I Novelobject recognition test. All the data are expressed as mean ± SEM (n = 10 per group). ***p < 0.001 versus the CON group. #p < 0.05, ##p < 0.01,###
p < 0.001 versus the CSDS group. &p < 0.05, &&p < 0.01 versus the CSDS + EDA group

Dang et al. Journal of Neuroinflammation

Fig. 16 (See legend on previous page.)(2022) 19:41

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Dang et al. Journal of Neuroinflammation(2022) 19:41

Page 25 of 29

Fig. 17 Schematic illustration of the proposed mechanisms underlying EDA-related antidepressant and anxiolytic efficacy

reduced in the blood of MDD patients compared withhealthy subjects [84]. Moreover, the Sirt1 activity indentate gyrus (DG) of Hip was decreased when chronic
stress exposure and genetic or pharmacologic ablationof Hip Sirt1 can lead to depression [85]. Nrf2, an important antioxidant, is an important downstream target of
Sirt1 and plays a crucial role in improving the resistanceto oxidative stress damage. Previous studies showed that
Nrf2 knockout mice exhibited depressive-like behavior
and CUMS model decreased Nrf2 expression in the rat
Hip [86, 87]. Besides, corticosterone treated mice showeda lower level of Nrf2 protein expression in the cortex and
Hip [88]. Recently, accumulating evidence revealed thata close link between Nrf2/HO-1 pathway and MDD [88,89]. It is mentioned above that we found the difference inglutathione metabolism via KEGG pathway enrichment.
Interestingly, the gene of Gpx4 plays a key role in glutathione metabolism and Nrf2 can directly or indirectly
regulate Gpx4 protein expression and function [90].

Therefore, we speculated that EDA improved depressive and anxiety-like behaviors by activating the Sirt1/
Nrf2/HO-1/Gpx4 signaling pathway. Here, we observedthat mice underwent CSDS exposure displayed reduced
protein expressions of Sirt1, Nrf2, HO-1 and Gpx4 in the
Hip and Sirt1 in the mPFC, which were all elevated by
EDA intervention. The reasons for the divergent results
between the Hip and mPFC are unclear, but one possibility may be due to different neurons having different levelsof vulnerability to OS [91]. Previous studies reported that
Hip was the most susceptible region to OS. It is known
that the dentate gyrus (DG–cornu ammonis 3(CA3)showed structural plasticity. Mounting studies showedthat pyramidal cells in CA3 and granule cells in DG are
OS prone regions, whereas other literature reported thatpyramidal neurons in CA1 are more susceptible to oxidative stress. It was reported that oxidative damage of
DG-CA1/3 function can reduce neuron proliferation,
alter neuronal plasticity and disrupt neurogenesis. On theother hand, the frontal cortex seems to be more resilient

Dang et al. Journal of Neuroinflammation(2022) 19:41

to OS [71]. Chronic stress stimuli may cause dendritic
shrinking and alter neuronal connectivity within mPFC
[91].
Interestingly, mRNA levels of Nrf2, HO-1 in the Hip
and mPFC and Sirt1 in the mPFC increased, while its
protein levels in the Hip were down-regulated. We
deduced this disparity between mRNA and protein levels suggests that post-transcriptional regulation, translational efficiency, and post-translational modifications
alter protein levels. One possible explanation is that
reduced translational efficiency may be compensatedby increased transcriptional activity [92]. However, thedetailed mechanism is not clear.
Ferroptosis is a recently identified type of regulated
cell death and has also been reported to be associatedwith several neurological diseases, including neurodegeneration, stoke and neurotrauma [93]. However, there
is still lack of reports about ferroptosis in the psychiatric diseases, especially depression and anxiety. First, we
employed TEM to examine the ultrastructure of neurons and microglia in the Hip and mPFC. Surprisingly,
we found the most prominent characteristic of ferroptosis, reduction volume, ruptured mitochondria in the
hippocampal microglia of CSDS exposed mice. Consistent with the shrunken mitochondria observed in
hippocampal microglia, CSDS-induced mice had lower
SOD, GSH-PX and T-AOC levels, as well as higher GSH
level in the hippocampal tissue. Furthermore, our results
showed that the expression of ferroptosis-related proteins, including Gpx4 and Nrf2 were down-regulated in
the Hip of CSDS exposed mice. Intriguingly, these effects
were all reversed with EDA treatment. Taken together,
these results inferred that ferroptosis could play a rolein depression and anxiety and EDA can relieve CSDSinduced ferroptosis.
Since Gpx4 was identified as a key regulatory factor in
ferroptosis and the protein expression of Gpx4 changed
after CSDS exposure in the Hip, but not in the mPFC.
Hence, we then used Gpx4 knockdown virus in the Hip
to explore the role of Gpx4-related ferroptosis in theeffects of EDA. Strikingly, EDA-induced antidepressantand anxiolytic effects were abolished. Unexpectedly, theobject recognition memory was impaired after AAV–EGFP–Gpx4–miRNAi injection, indicating that Gpx4
may be associated with memory impairment.
Nevertheless, our study is not without limitations.
First, the pathway from Sirt1 to Gpx4 is complex and wetargeted only one portion of this pathway. Second, EDA
alleviated neuroinflammation and neuronal loss in boththe Hip and mPFC regions. Hence, we could not rule out
the possibility that other mechanisms in the mPFC mayunderlie the functions of EDA. Third, although we demonstrated Gpx4 plays a key role in the effects of EDA,

Page 26 of 29

further research is required to investigate the underlying
mechanisms of Gpx4-related ferroptosis in depression
and anxiety.

Conclusion
In summary, our study demonstrates for the first timethat EDA could ameliorate depressive and anxietylike behaviors, OS and neuroinflammation in a CSDSinduced mice model of depression. The underlying
molecular mechanism may involve Gpx4-mediated ferroptosis via Sirt1/Nrf2/HO-1 pathway.
These results also highlight aberrant expression of
Gpx4 as a potential mechanism in depression and suggestthat Gpx4 mediated ferroptosis may be a promising new
target for the treatment of MDD.
Abbreviations
OS: Oxidative stress; EDA: Edaravone; CSDS: Chronic social defeat stress; MDD:
Major depression disorder; SI: Social interaction; SPT: Sucrose preference test;
OFT: Open field test; EPM: Elevated plus maze; NOR: Novel object recognition;
TST: Tail suspension test; FST: Forced swim test; TEM: Transmission electron
microscopy; WB: Western blotting; qRT-PCR: Quantitative real-time polymerase
chain reaction; AAV: Adeno-associated virus; Hip: Hippocampus; mPFC: Medial
prefrontal cortex; RNS: Reactive nitrogen species; LTP: Long-term potentiation;
Sirt1: Silent information regulator 2 homolog 1; Nrf2: Nuclear factor erythroid
2-related factor 2; HO-1: Heme oxygenase-1; Gpx4: Glutathione peroxidase 4;
FDA: Food and Drug Administration; ALS: Amyotrophic lateral sclerosis; CON:
Control; IZ: Interaction zone; MDA: Malondialdehyde; SOD: Superoxide dismutase; GSH: Glutathione; ANOVA: Analysis of variance; IPA: Ingenuity Pathway
Analysis; AMP: Adenosine monophosphate; GMP: Guanosine monophosphate;
GDP: Guanosine diphosphate; ADP: Adenosine diphosphate; KEGG: Kyoto
Encyclopedia of Genes and Genomes; CUMS: Chronic unpredictable mild
stress; BBB: Blood-brain barrier; GFAP: Glial fibrillary acidic protein; Iba-1: Ionized calcium-binding adaptor molecule 1; PBS: Phosphate-buffered solution;
PC: Passive coping; AD: Alzheimer’s disease; BSA: Bovine serum albumin; CA3:
Cornu ammonis 3; CX30: Connexin 30; CX43: Connexin 43; DG: Dentate gyrus;
ELISA: Enzyme-linked immunosorbent assay; 4-HNE: 4-hydroxynonenal; NADP:
Nicotinamide adenine dinucleotide phosphate; 8-OH-dG: 8-hydroxydeoxyguanosine; ROS: Reactive oxygen species; T-AOC: Total antioxidant capacity;
TBA: Thiobarbituric acid; TBARS: Total antioxidant capacity; TREM2: Triggering
receptor expressed on myeloid cells 2.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​022-​02400-6.
Additional file 1: Figure S1. Ultrastructure of microglia in the CSDS
model of Hip and mPFC with EDA treatment. a Electron micrographs
showed mitochondrial damages in the hippocampal microglia (redarrows). Scale bars, 2 μm (upper panel) and 500 nm (lower panel). b
Electron micrographs showed mitochondrial damages in the mPFC microglia (red arrows). Scale bars, 2 μm (upper panel) and 500 nm (lower panel).
Additional file 2: Figure S2. Pathway analysis of metabolomic alternations induced by CSDS and EDA treatment. IPA was conducted to identify
the top canonical signaling pathways affected in CSDS mice with orwithout concomitant EDA treatment.
Additional file 3: Figure S3. Effect of EDA on Sirt1/Nrf2/HO-1/Gpx4 pathway in mRNA expression levels. a–h mRNA expression of Sirt1 (a), Nrf2 (b),HO-1 (c) and Gpx4 (d) in the hippocampal region. e–h mRNA expressionof Sirt1 (e), Nrf2 (f), HO-1 (g) and Gpx4 (h) in the mPFC region. Data areDang et al. Journal of Neuroinflammation(2022) 19:41

presented as mean ± SEM (n = 8 per group). *p < 0.05, **p < 0.01 versus
the CON + Vehicle group. #p < 0.05, ###p < 0.001 versus the CSDS + Vehicle
group.

Page 27 of 29

3.

4.
Acknowledgements
Not applicable.
Authors’ contributions
PX and RZD designed the experiments; RZD, MYW and XHL performed theexperiments; HYW, LXL, QYW, and JTZ assisted with experiments; RZD and
XHL analyzed the data; RZD wrote the manuscript; PJ, LMZ, JL and PX revised
the manuscript. The authors read and approved the final manuscript.
Funding
This work was supported by the National Key Research and Development
Program of China (Grant No. 2017YFA0505700), the Non-Profit Central
Research Institute Fund of Chinese Academy of Medical Sciences (Grant No.
2019PT320002), and the Natural Science Foundation Project of China (Grant
No. 81820108015).
Availability of data and materials
All data generated in this study are included in this manuscript.

Declarations
Ethics approval and consent to participate
All experimental procedures were conducted in accordance with the Ethics
Committee of Chongqing Medical University.

5.
6.

7.

8.
9.

10.

11.

Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
NHC Key Laboratory of Diagnosis and Treatment on Brain Functional
Diseases, The First Affiliated Hospital of Chongqing Medical University, 1
Youyi Road, Yuzhong District, Chongqing 400016, China. 2 Departmentof Neurology, The First Affiliated Hospital of Chongqing Medical University,
Chongqing 400016, China. 3 College of Stomatology and Affiliated Stomatological Hospital of Chongqing Medical University, Chongqing 401147, China.
4
Department of Neurology, Yongchuan Hospital of Chongqing Medical
University, Chongqing 402160, China. 5 Department of Neurology, Chongqing
University Three Gorges Hospital, Chongqing 404100, China. 6 Departmentof Neurology, Xinxiang Central Hospital, Xinxiang 453000, China. 7 Chongqing
Key Laboratory of Oral Diseases and Biomedical Sciences, College of Stomatology and Affiliated Stomatological Hospital of Chongqing Medical University,
Chongqing 401147, China. 8 Key Laboratory of Psychoseomadsy, Stomatological Hospital of Chongqing Medical University, Chongqing 401147, China.
9
Department of Neurology, First Affiliated Hospital of Kunming Medical
University, Kunming 650032, China. 10 Department of Psychiatry and Behavioral
Sciences, College of Medicine, SUNY Upstate Medical University, Syracuse, NY,
USA.

12.

13.

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15.

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18.

Received: 27 August 2021 Accepted: 24 January 2022
19.

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