(2024) 21:132
Yang et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03121-8

Journal of Neuroinflammation

Open Access

RESEARCH

Protecting effects of 4‑octyl itaconate
on neonatal hypoxic‑ischemic encephalopathy
via Nrf2 pathway in astrocytes
Yanping Yang1, Yang Li1, Wenyi Yang1, Xueying Yang2, Man Luo3, Ling Qin2* and Junchao Zhu1*

Abstract
Background Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the most common neurological problems
occurring in the perinatal period. However, there still is not a promising approach to reduce long-term neurodevelopmental outcomes of HIE. Recently, itaconate has been found to exhibit anti-oxidative and anti-inflammatory effects.
However, the therapeutic efficacy of itaconate in HIE remains inconclusive. Therefore, this study attempts to explore
the pathophysiological mechanisms of oxidative stress and inflammatory responses in HIE as well as the potential
therapeutic role of a derivative of itaconate, 4-octyl itaconate (4OI).
Methods We used 7-day-old mice to induce hypoxic-ischemic (HI) model by right common carotid artery ligation
followed by 1 h of hypoxia. Behavioral experiments including the Y-maze and novel object recognition test were
performed on HI mice at P60 to evaluate long-term neurodevelopmental outcomes. We employed an approach combining non-targeted metabolomics with transcriptomics to screen alterations in metabolic profiles and gene expression in the hippocampal tissue of the mice at 8 h after hypoxia. Immunofluorescence staining and RT-PCR were used
to evaluate the pathological changes in brain tissue cells and the expression of mRNA and proteins. 4OI was intraperitoneally injected into HI model mice to assess its anti-inflammatory and antioxidant effects. BV2 and C8D1A cells were
cultured in vitro to study the effect of 4OI on the expression and nuclear translocation of Nrf2. We also used Nrf2siRNA to further validate 4OI-induced Nrf2 pathway in astrocytes.
Results We found that in the acute phase of HI, there was an accumulation of pyruvate and lactate in the hippocampal tissue, accompanied by oxidative stress and pro-inflammatory, as well as increased expression of antioxidative
stress and anti-inflammatory genes. Treatment of 4OI could inhibit activation and proliferation of microglial cells
and astrocytes, reduce neuronal death and relieve cognitive dysfunction in HI mice. Furthermore, 4OI enhanced
nuclear factor erythroid-2-related factor (Nfe2l2; Nrf2) expression and nuclear translocation in astrocytes, reduced proinflammatory cytokine production, and increased antioxidant enzyme expression.
Conclusion Our study demonstrates that 4OI has a potential therapeutic effect on neuronal damage and cognitive deficits in HIE, potentially through the modulation of inflammation and oxidative stress pathways by Nrf2
in astrocytes.
Keywords Hypoxia–ischemia, Itaconate, Nrf2, Astrocyte, Cognitive dysfunction*Correspondence:
Ling Qin
qinlingling@yahoo.com
Junchao Zhu
zhujunchao1@hotmail.com
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Yang et al. Journal of Neuroinflammation(2024) 21:132

Introduction
Neonatal hypoxic-ischemic encephalopathy (HIE) is abrain injury caused by perinatal asphyxia in neonates,with its incidence from 1 to 8 per 1000 live births indeveloped countries and reaching as high as 26 per 1000
live births in underdeveloped countries [1–3]. Currently,
therapeutic hypothermia (TH) is the only intervention
approved for the treatment of HIE [4]. However, its application is constrained by a narrow therapeutic window,
requiring initiation within six hours post-birth. Despite
the widespread implementation of TH in high-resource
settings, approximately 30–50% of infants with moderate-to-severe HIE treated with TH still suffer from mortality or substantial neurological impairments, such ascerebral palsy, epilepsy and cognitive dysfunction [5–10].
Therefore, it is imperative to explore new neuroprotective therapies to reduce the mortality rate of HIE patientsand improve their neurological development outcomes.
During the initial stage of HIE pathophysiology (0–6 h),
the brain undergoes a complex cascade of processes associated with energy depletion, including the accumulationof excitotoxic substances, oxidative stress, and intracellular acidosis [11–13]. Compared to the adult brain, theneonatal brain has a high level of mitochondrial respiration for oxygen consumption and low concentrations ofantioxidants, rendering it particularly susceptible to thedetrimental effects of oxidative stress [14, 15]. Previous
studies have indicated that the hippocampus is more susceptible to oxidative stress damage induced by hypoxia–
ischemia due to its high content of polyunsaturated fatty
acids and abundance of transition metals such as ironand copper, leading to adverse cognitive effects [16–18].
Although studies have shown that the hippocampus
is a brain region with neurogenesis and neurogenesis
increases after HI injury, it is still impossible to fully compensate for the neurological impairment after HI [19, 20].
Therefore, it is imperative to study the pathophysiologicaland molecular mechanisms of HI damage to hippocampal neurons, in order to provide experimental evidence
for the development of new therapeutic approaches.
Following HI, damaged neurons activate brain innate
immune cells-microglia through multiple pathways
such as damage-related molecular patterns (DAMPs)
by releasing ADP, heat shock protein and other substances, prompting them to release inflammatory factors such as IL-1β, IL-6, and TNF-α, as well as reactive
oxygen species, and induce neuronal damage and evendeath through pathways such as NF-κB [21]. Studies
have shown that activated microglia release CSF-1, which
promotes the transformation of local astrocytes fromnutrient-supportive to reactive state, and induces the
production of pro-inflammatory cytokines. This crosstalk between microglia and astrocyte would lead to a

Page 2 of 21

persistent inflammatory response [22]. Oxidative stressinduced inflammatory responses, if persistent, result inwidespread neuronal death and permanent neurologicalimpairment in the hippocampus [23, 24].
Therefore, it is crucial to identify key molecular mechanisms of neuron-glial cross-talk induced by oxidative
stress and inflammatory response in hippocampus. The
results of this study can provide a basis for targeted therapy for cognitive dysfunction in HIE. To achieve this, In
this study, we employed an approach combining nontargeted metabolomics with transcriptomics, to screen
alterations in metabolic profiles and gene expression
in the hippocampal tissue of a mouse model 8 h after
hypoxia–ischemia (HI). We identified significant metabolic perturbations, including the accumulation of lactate
and pyruvate. And the RNA-sequence (RNA-seq) results
showed high expression of inflammatory genes related
to IFN and nuclear factor (NF)- κB pathways, as well as
elevated expression of antioxidant stress genes, such asactivating transcription factor 3 (Atf3), the nuclear factor erythroid-2-related factor (Nfe2l2; Nrf2), and heme
oxygenase 1 (Hmox-1;HO-1). The results of metabolomic
and RNA-Seq indicate that pro-inflammatory and antiinflammatory, oxidative and antioxidative pathways were
simultaneously activated.
Based on these mechanistic insights, we speculate that
potentiating Nrf2- mediated anti-inflammatory and antioxidative pathways may have a protective effect on HIE.
Recently, some studies have shown that 4-octyl itaconate(4OI), a derivative of itaconate, could facilitate Nrf2 totranslocate into the nucleus and upregulate the expression of anti-inflammatory and antioxidant genes [25–28].
In addition, potential therapeutic effects of itaconate has
been confirmed in various central nervous system disorders, including Alzheimer’s disease and spinal cord injury[29–32]. However, the role and mechanism of 4OI in HI
remain unexplored. Therefore, we injected 4OI intraperitoneally into HI model mice, it was found that 4OI exerts
neuroprotective effects in HI mice through increasing the level of Nrf2 in astrocytes, thereby suppressing
the production of pro-inflammatory cytokines, such as
interleukin (IL)-1β and IL-6, and regulating the expression of antioxidant enzymes HO-1. Our findings provide
experimental evidence for the essential role of Nrf2 in thepathological process of HIE, and the potential for clinicalapplications of 4OI as a therapeutic drug to HIE.

Materials and methods
Animals

In this study, all procedures and protocols were approvedby the Animal Care and Use Committee of China Medical University. Male and female C57BL/6 wild-type mice
aged P7–P60 weeks were obtained from the Department

Yang et al. Journal of Neuroinflammation(2024) 21:132

of Laboratory Animal Science of China Medical University. The pups, along with their mothers, were bred andreared under standard conditions with a 12 h/12 h light/
dark cycle, at an environmental temperature of 22 ± 1 °C
and a humidity of 30%—50%, with ad libitum access tofood and water. All surgical procedures were conductedunder anesthesia to minimize animal suffering. To reduce
experimental bias and ensure unbiased results, all animals were randomly assigned to groups generated by
Excel. All researchers involved in neurological testing
and molecular experiments were blinded. Each intervention group included an appropriate control group.
Neonatal hypoxia–ischemia model and drug
administration.

This study utilized the classic model of neonatal hypoxicischemic brain damage [33], conducting experiments inline with the animal ethics guidelines set by China Medical University. We used 3–5 g of mixed gender (randomized males and females) P7 pups for experiments.
Initially, the pups were anesthetized with 2% isoflurane.
Following anesthesia, their neck skin was disinfectedwith alcohol. Subsequently, a 1 cm midline incision wasmade on the right anterior neck using a scalpel. A gentle
blunt dissection was then performed, delicately separating the right common carotid artery from surrounding
structures. Right common carotid artery was carefully
double ligated with 8–0 surgical silk and subsequently
cut between the ligatures. After this, the skin was sutured
with thread. To maintain sterility, the entire surgical process was conducted under aseptic conditions, ensuringthat each surgery lasted no more than 10 min. Postoperatively, the pups were placed on a 37 °C heating blanket for
1 h for recovery. In our study, we modified a sealed miceanesthesia chamber, with one end connected to purenitrogen and the other to pure oxygen. The oxygen concentration could be adjusted by altering the flow rate ofoxygen or nitrogen through a gas valve. Additionally, thechamber was equipped with a CY-12 oxygen analyzer anda thermometer for real-time monitoring of oxygen concentration and temperature. The pups were placed in atemperature-controlled hypoxic chamber for 1 h (7% O­ 2,
93% ­N2, at 37 °C). Finally, the pups were returned to theirmother and monitored daily. In contrast, sham-operated
mice only underwent a neck skin incision without any
ligation or hypoxia. Due to the absence of a monitoring
system for vital signs during the modeling process, there
was an unavoidable mortality rate of approximately 5%
among the neonatal mice.
In determining the dosage and duration for 4OI treatment, we referenced a recent review on the dosage of
4OI (PMID: 34055739), which provided a foundation for
selecting the doses of 10, 25, and 50 mg/kg. Additionally,

Page 3 of 21

we decided to administer 4OI at the determined optimaldose every 24 h for a total of 3 days, based on studies
indicating that delayed neuronal death in the hippocampus occurs 3–4 days following ischemia–reperfusion(PMID: 31251935).
The OGD/R model

BV2 microglial cells and C8-D1A astrocytes were purchased from Procell (Wuhan, China). Both cell types
were cultured in complete medium containing 10% fetal
bovine serum for two days. At the onset of the oxygen–glucose deprivation (OGD) experiment, the culture
medium was replaced with a glucose- and serum-freecomplete medium, and cells were placed in a hypoxic
chamber (1% ­O2, 5%CO2, 94%N2, at 37 °C, Biospherix
C21, American) for 4 h of hypoxia. Following the OGD/R,
the medium was again replaced with serum-containing
complete medium, and cells were further cultured for
12 h to complete the oxygen–glucose deprivation/reoxygenation (OGD/R) process. In contrast, control groupcells were cultured in a standard incubator (5% ­CO2, 94%­O2, at 37 °C) with complete medium. The experiment was
divided into three groups: control (Control), OGD/R, and
OGD/R + 4OI. In the control and OGD/R groups, DMSO
was added to the culture medium to a final concentrationof 0.2%. In the OGD/R + 4OI group, 4OI (50 μM, 0.1%
DMSO) was dissolved in the culture medium. After the
OGD treatment, cell supernatants were collected for subsequent analyses.
In vitro cell transfection

Small interfering RNA (siRNA) targeting Nrf2 (si-Nrf2),
and negative scramble control siRNA (si-Negative) werepurchased from Thermo Fisher Scientific (USA). C8-D1A
astrocytes were cultured in complete medium in 6-wellplates for 48 h, and then transfected with either Nrf2
siRNA or Nrf2-negative siRNA using Lipofectamine
RNAi MAX (Invitrogen, Waltham, MA, USA). After
24 h, the transfected cells were exposed to OGD/R for 4 h
before Western blotting and further analysis.
Western Blot

Western blotting was performed in accordance with the
manufacturer’s specifications. After OGD treatment,
C8-D1A and BV2 cells were collected. Cells were lysed
in RIPA buffer containing phenylmethylsulfonyl fluoride(Santa Cruz, CA, USA) for protein extraction, followedby centrifugation at 12,000 rpm for 15 min at 4 °C toremove insolubles. Protein concentration was quantified
using the BCA Protein Assay Kit (Beyotime technology,
China). Equal amounts of protein were loaded onto SDSPAGE gels for electrophoresis, followed by transfer topolyvinylidene fluoride membranes. After blocking with

Yang et al. Journal of Neuroinflammation(2024) 21:132

Page 4 of 21

5% non-fat milk for 2 h, the membranes were incubatedovernight at 4 °C with Nrf2, IL-1β (1:2000, 16,806–1AP, Protrintech) and HO-1 (1:1000, 10,701–1-AP, Protrintech) antibody. After primary antibody incubation,
membranes were washed at least three times with TBS
containing 0.2% Tween-20, then incubated with theappropriate secondary antibody at room temperaturefor 1.5 h. The membranes were then washed three times
with TBST. Finally, detection of protein bands was conducted using an enhanced chemiluminescence reagent
and imaged with an imaging densitometer. Data analysis
was performed using ImageJ software.

slices were washed three times with PBS solution (5 min
per wash) and incubated with secondary antibodies,including CoraLite488 (1:200, SA00013-2, Proteintech,USA) and CoraLite594 (1:200, SA00013-4, Proteintech,USA), at room temperature for 2 h. After washing with
PBS, sections were covered with DAPI (SL1841, Coolaber, China). Finally, the slides were observed using a fluorescence microscope (BX53, Olympus, Japan), and thenumber of cells in the hippocampus were automatically
identified and counted using IMARIS software (Bitplane,
Switzerland).

TTC staining

At P7, mice were deeply anesthetized with 3% isoflurane
and were perfused transcardially with 100 mL 0.9% NaCl
(4 °C). After the perfusion, hippocampal tissues from theside corresponding to the ligated right common carotid
artery were harvested and immediately immediately
preserved in the RNA later solution. Total RNAs wereextracted from hippocampus of sham and HI mice using
TRIzol Reagent (Invitrogen, cat. NO 15596026) following
the methods by Chomczynski et al.[37]. DNA digestion
was carried out after RNA extraction by DNaseI. RNA
quality was determined by Nanodrop TM OneC spectrophotometer (Thermo Fisher Scientific Inc). RNA Integrity was confirmed by 1.5% agarose gel electrophoresis.
Qualified RNAs were finally quantified by Qubit3.0 with
QubitTM RNA Broad Range Assay kit (Life Technologies, Q10210).
2 μg total RNAs were used for stranded RNA sequencing library preparation using KC-DigitalTM StrandedmRNA Library Prep Kit for Illumina® (Catalog NO.
DR08502, Wuhan Seqhealth Co., Ltd. China) following
the manufacturer’s instruction. The kit eliminates duplication bias in PCR and sequencing steps, by using uniquemolecular identifier (UMI) of 8 random bases to label
the pre-amplified cDNA molecules. The library products
corresponding to 200–500 bps were enriched, quantified
and finally sequenced on DNBSEQ-T7 sequencer (MGI
Tech Co., Ltd. China) with PE150 model.

The pups were fully anesthetized with isoflurane (3%) andperfused with PBS through the heart to collect the brain
24 h post-HI. The brain was then frozen in a -20℃ freezer
for 20 min, and sliced into 3 mm coronal sections with ablade. A total of 4 coronal brain slices were prepared. The
brain slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining solution at
37℃ in the dark for 30 min, and then fixed with 4% paraformaldehyde [34]. The brain infarct volume of the micewas calculated using the Image-J software (version) by
scanning the coronal brain tissue sections with a scanner (Hp laserJet Pro MFP M427dw). The formula used for
calculating the infarct volume (%) was as follows: Infarct
volume (%) = [Volume of the contralateral hemisphere—(Volume of the ipsilateral hemisphere—infarct volume)] /Volume of the contralateral hemisphere × 100% [35].
Evaluation of brain tissue loss

The percentage of brain tissue loss = (contralateral hemisphere – ipsilateral hemisphere)/contralateral hemisphere × 100% [36].
Immunofluorescence staining

The mice were anesthetized at P8, P14 and P60, and thenperfused transcardially with ice-cold PBS and 4% paraformaldehyde. The brains were fixed with 4% paraformaldehyde for 24 h and transferred to 30% sucrose solution
for dehydration for three days. The brain tissues weresliced continuously at -20 °C using a freezing microtome(Minux, FS800, RWD) to obtain coronal sections of
15 μm-thick coronal sections. The sectioned tissue wasthen fixed on glass slides and used for immunofluorescence staining. The brain slices were washed threetimes with PBS solution (5 min per wash), blocked with
10% goat serum for 2 h, and then incubated with primary antibodies, including Anti-Neun (1:500, ab177487,
Abcam, USA), Anti-GFAP (1:500, ab7260, Abcam, USA),
Anti-Iba1 (1:200, ab178847, Abcam, USA), and Anti-Nrf2(1:50, 1639–1-AP, Proteintech, USA), at 4 °C for 12 h. The

RNA extraction, library preparation and sequencing

RNA‑Seq data analysis

Raw sequencing data was first filtered by Trimmomatic(version 0.36), low-quality reads were discarded andthe reads contaminated with adaptor sequences were
trimmed. Clean Reads were further treated with inhouse scripts to eliminate duplication bias introduced inlibrary preparation and sequencing. In brief, clean reads
were first clustered according to the UMI sequences, inwhich reads with the same UMI sequence were grouped
into the same cluster. Reads in the same cluster werecompared to each other by pairwise alignment, and thenreads with sequence identity over 95% were extracted toYang et al. Journal of Neuroinflammation(2024) 21:132

a new sub-cluster. After all sub-clusters were generated,
multiple sequence alignment was performed to get one
consensus sequence for each sub-clusters. After thesesteps, any errors and biases introduced by PCR amplification or sequencing were eliminated.
Reads mapped to the exon regions of each gene werecounted by featureCounts (Subread-1.5.1; Bioconductor) and then RPKM was calculated. Genes differentiallyexpressed between groups were identified using theedgeR package (version 3.12.1). A p-value cutoff of 0.05
and Fold-change cutoff of 2 were used to judge the statistical significance of gene expression differences. Gene
ontology (GO) analysis and Kyoto encyclopedia of genes
and genomes (KEGG) enrichment analysis for differentially expressed genes were both implemented by KOBAS
software (version: 2.1.1) with a P-value cutoff of 0.05 tojudge statistically significant enrichment. Alternative
splicing events were detected by using rMATS (version
3.2.5) with a FDR value cutoff of 0.05 and an absolutevalue of Δψ of 0.05.
Sample preparation and extraction

The sham and HI hippocampal sample stored at -80 °C
refrigerator was thawed on ice and vortexed for 10 s. 50
μL of sample and 300 μL of extraction solution (ACN:
Methanol = 1:4, V/V) containing internal standards
were added into a 2 mL microcentrifugetube. A 180 μL
aliquots of supernatant were transferred for LC–MS
analysis. The original data file acquisited by LC–MS wasconverted into mzML format by ProteoWizard software.
Peak extraction, peak alignment and retention time correction were respectively performed by XCMS program.
The “SVR” method was used to correct the peak area. The
peaks with detetion rate lower than 50% in each group of
samples were discarded. After that, metabolic identification information was obtained by searching the laboratory’s self-built database, integrated public database, AI
database and metDNA.
Metabolomics analysis

Unsupervised PCA (principal component analysis)
was performed by statistics function prcomp withinR(www.r-​proje​ct.​org). The data was unit variance scaled
before unsupervised PCA. The HCA (hierarchical cluster analysis) results of samples and metabolites were
presented as heatmaps with dendrograms, while pearson correlation coefficients (PCC) between samples
were caculated by the cor function in R and presented
as only heatmaps. Both HCA and PCC were carriedout by R package ComplexHeatmap. For HCA, normalized signal intensities of metabolites (unit variance scaling) are visualized as a color spectrum. For two-group
analysis, differential metabolites were determined byPage 5 of 21

variable importance on projection (VIP) value > 1 andP-value (P-value < 0.05, Student’s t test). VIP values wereextracted from OPLS-DA result, which also contain scoreplots and permutation plots, was generated using R package MetaboAnalystR. The data was log transform (log2)
and mean centering before OPLS-DA. In order to avoid
overfitting, a permutation test (200 permutations) was
performed. Identified metabolites were annotated using
KEGG Compound database (http://​www.​kegg.​jp/​kegg/​compo​und/), annotated metabolites were then mapped
to KEGG Pathway database (http://​www.​kegg.​jp/​kegg/​
pathw​ay.​html). Significantly enriched pathways are identified with a hypergeometric test’s P-value for a given listof metabolites.
Real‑time PCR

After completely anesthetizing the mice with isoflurane,
the hippocampal tissue on the same side was collected.
The RNA was extracted using an RNA Isolation KitBOX2 (RC101-01, Vazyme, China) following the manufacturer’s instructions, and its concentration and quality
were measured using a fluorescence method. Reverse
transcription was performed using a cDNA synthesis kit(TaKaRa Biotechnology). IL-1β and IL-6 primers (Sangon Biotech, Shanghai, China) were used for qPCR on
a SYBR-Green pre-mix (TransGen Biotech). The cycle
parameters of the CFX96 sequence detection systemwere 95 °C for 5 min, followed by 40 cycles of 95 °C for
15 s, 58 °C for 30 s, and 72 °C for 20 s. Finally, the mRNAlevel of β-actin was used as an internal control to standardize the expression level of the target gene. The ΔΔCt
values of each group were analyzed, and the mRNA
expression of different groups was normalized to 2-ΔΔCt(Table 1).
Behavior tests
Negative geotaxis

Set the wooden board at a 45-degree angle and release
the mouse with its head facing the lower end of the slope
in the middle of the board. Record the time it takes forthe mouse to turn its head and body 180 degrees, repeating the experiment three times per mouse and calculating
the average. If the mouse does not turn its head within
60 s, the maximum value is recorded as 60 s.
Righting reflex test

Place the young mouse with its back facing upwards on awooden board, ensuring that its head and back are tightly
against the board before releasing it. Subsequently,
record the time it takes for the young mouse to turn froma supine position to a prone position with its limbs facing the board. Repeat this process three times for eachmouse and calculate the average time. If a mouse fails toYang et al. Journal of Neuroinflammation(2024) 21:132

Table 1 Primers used in this study
Primer Forward sequence (5’-3’)

Reverse sequence (5’-3’)

IL-1β

CGT​C TC​CCA​GAG​CCA​ATC​C

CAC​CAG​GCT​GAC​T TT​GAG​
TGAGT​

IL-6

ATC​CAG​T TG​CCT​TCT​TGG​GAC​TGA​TAA​GCC​TCC​GAC​T TG​TGA​AGT​
GGT​

C3

TTC​C TT​CAC​TAT​GGG​ACC​AGC​

TCC​T TA​C TG​GCT​GGA​ATC​T TGA​

Ctla2aGGT​GAG​AGC​AGA​GAA​AAG​CA

CTG​TGT​C TT​TCT​TCA​T TC​AGA​
TTG​T

Ifi211

AGC​TGA​T TC​TGG​ATT​GGG​CATCA​CAC​ACT​T TC​TGC​GTG​C T

Nfe2l2

ACT​ACA​GTC​CCA​GCA​GAG​TGAT​TCA​CAC​ACT​T TC​TGC​GTG​C T

Atf3

TAC​CGT​CAA​CAA​CAG​ACC​CC

CCA​GTT​TCT​C TG​ACT​C TT​TCTGC​

HO-1

CCG​C TA​CCT​GGG​TGA​CCT​C TC​

CCT​C TG​ACG​AAG​TGA​CGC​CATC​

β-actin

TAA​AGA​CCT​C TA​TGC​CAA​CAC​
AGT​

CAC​GAT​GGA​GGG​GCC​GGA​
CTC​ATC​

IL interleukin, C3 complement component 3, Ctla2a cytotoxic T lymphocyteassociated protein 2 alpha, Ifi211 interferon activated gene 211, Nfe2l2 nuclear
factor, erythroid derived 2, like 2, Atf3 activating transcription factor 3, HO-1
heme oxygenase 1

turn over within 60 s, record the time as the maximum
value of 60 s.
Y‑mazeThe Y-maze consists of three arms, each measuring
10 cm × 40 cm × 15 cm, with an angle of 120° between
each arm. The entire Y-maze experiment consists of twophases. In the first phase, one arm of the Y-maze (thenovel arm) is randomly blocked using an opaque barrier, and the mouse is placed randomly in one of theremaining two arms (the start arm) for free explorationfor 5 min. Afterwards, the mouse is returned to the home
cage. The Y-maze is cleaned of feces and wiped with 75%
ethanol to eliminate any olfactory cues that may affect themouse. The second phase of the experiment begins 5 minlater, during which the opaque barrier is removed, andthe mouse is placed back in the same start arm for free
exploration for 5 min while being monitored by a camera. A custom-built software is used to track the mouse’s
location and analyze its trajectory after the experiment.
The exploration time of the novel arm is recorded, andthe percentage of time spent exploring the novel arm is
calculated as a proportion of the total exploration time.
Novel object recognition (NORT)The novel object recognition test was used to detect
short-term memory function. The experiment consists ofthree phases: habituation, training, and testing. During
the habituation phase, the mouse is allowed to adapt to a
black and white box (50 cm × 40 cm × 30 cm) for 30 min.
The training phase begins 24 h later, during which themouse is placed in the box with two identical objects

Page 6 of 21

for free exploration for 10 min, followed by returning
the mouse to its home cage. The Y-maze is cleaned offeces and wiped with 75% ethanol to eliminate any olfactory cues that may affect the mouse. The testing phasestarts by replacing one object with a novel object, andthe mouse is allowed to explore for another 10 min. The
mouse’s movement trajectory is recorded and analyzed
by a custom-built software to quantify the exploration
time on different objects and evaluate the cognitive function of the mouse using the novel object recognition test
index. The discrimination index (DI) is calculated as follows: DI = [(novel object exploration time)—(familiar
object exploration time)] / [(novel object explorationtime) + (familiar object exploration time)], where DI represents the recognition ability of the mouse [38].
The open field test (OFT)The open field apparatus consisted of a rectangular behavioral box made of white plastic panels(54 cm × 40 cm), with a height of 30 cm. Prior to theinitiation of the experiment, each mouse was placed in
the laboratory for 5 min in advance to acclimate to thelaboratory environment. At the start of the experiment,
mice were placed in the central area of the behavioral box
and allowed to freely move and explore the entire box for
30 min. Their activities were recorded by a camera (C270
HD Webcam, Logitech, Switzerland) positioned approximately 80 cm above the box. Following the test, mice
were removed from the behavioral box. Each animal was
subjected to the open field test only once. Between eachexperiment, excrement within the box was cleaned, andthe box was thoroughly disinfected with 75% ethanol toeliminate the impact of residual biological odors on theexperiment. Results include total distance traveled andduration in center (located in the center of arena with
25% of total area). The total duration of OFT, excluding
habituation, was 30-min.
The forced swimming test

The forced swim test is conducted in a cylindrical container filled with water, typically 30 cm in diameter and45 cm in height. The container is filled with water maintained at 22–25 °C, with a depth sufficient to preventthe mouse from touching the bottom with its feet or tailwhile preventing escape. Each mouse is gently placed
into the water and allowed to swim for a fixed duration,commonly 6 min. The initial 2 min serve as an acclimation period, and the subsequent 4 min are recorded foranalysis. The swim behaviors of the mice are captured
by a camera positioned above the container. Each mouseundergoes the forced swim test only once to prevent the
influence of repeated testing. Following the test, mice arecarefully removed from the water, dried, and returned toYang et al. Journal of Neuroinflammation(2024) 21:132

their home cages. The water in the container is changed
between each test to maintain cleanliness and consistent testing conditions. Immobility time was defined as
the time spent by the mouse floating in the water without
struggling and making only those movements necessary
to keep its head above the water.
The elevated O‑maze experiment

The elevated O-maze is a device developed from theelevated plus maze, designed to enable continuous exploration by experimental animals. The elevated O-mazeconsists of an elevated circular platform with an external
diameter of 50 cm and an internal diameter of 30 cm. It
is comprised of two opposite closed arms (10 cm wide
and 12 cm high) and two opposite open arms (10 cmwide), set at a height of 100 cm above the ground. Eachmouse was given 5 min to acclimate to the laboratoryenvironment before being gently lifted by the tail andplaced in an open arm of the elevated O-maze, where
it was allowed to explore for 10 min. The activity of themice in the elevated O-maze was captured by an overhead camera (C270 HD Webcam, Logitech, Switzerland).
Each experimental animal was subjected to the elevatedO-maze test only once. The entire elevated O-maze platform was cleaned with 75% ethanol between each experiment. The time spent in the open arms was used as anindex of anxiety-like behavior.
Statistical analysis

Statistical analysis in this study was performed using
GraphPad Prism 8 software (GraphPad Software, San
Diego, USA). The data were presented as mean ± standard error of the mean. Student’s t-test was used to analyze
significant differences between the two groups, and oneor two-way ANOVA was used to analyze the differencesmore than two groups, followed by Tukey’s post-hoc or
Sidak multivariate comparative analysis. A significancelevel of p < 0.05 was considered statistically significant.

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Results
Cognitive dysfunction in adult HI model mice

As shown in Fig. 1A, we used HI on 7-day-old mice as
an experimental model, which was the modified RiceVannucci neonatal hypoxia–ischemia model to ensure
the survival rate of pups [39]. The results of TTC staining showed that there were no white infarcts in the shamgroup. In the HI group, large white infarcts appeared in
the right cerebral hemisphere, and the average infarct
volume accounted for 55.08% ± 5.40% of the whole brain
volume (Fig. 1B, C). We performed several behavioral
tests on HI mice at P60 stage to evaluate long-term outcomes. Compared with the sham group, HI mice exhibited less time during exploration in the novel arm of the
Y-maze (Fig. 1D, E). The novel object recognition test
revealed that the discrimination index was significantly
lower in the HI group than in the sham group (Fig. 1F, G),
indicating the object recognition memory and the shortterm memory were impaired. Besides, no significant difference was found in open field, elevated O-maze andforced swimming tests (Fig. 1H–K). Our results showedthat HI caused cognitive dysfunction in mice.
Pathological Changes in the Hippocampus of Mice after HI

To find pathological change of hippocampus in HI mice,
we performed immunofluorescence staining and tissue
harvest at P8, P14 and P60 (Fig. 1A). The gross morphology of the brain in the HI group was similarly to that in
the sham group at P8 and P14, but smaller at P60. The
atrophy was especially significant in the right hippocampus (Fig. 2A). The HI group showed a clear decrease of
NeuN positive cells compared with the sham groupfrom P8-P14 (Fig. 2B, C). And HI mice exhibited obvious hippocampal atrophy at P60 (Fig. 2B). Iba1-positive
microglial cells in the sham group appeared mostly inthe resting ramified form with small round cell bodies
and thin processes, whereas those in HI group showed
considerable branch retraction, thickening, and amoebalike morphology from P8-P14 (Fig. 2D, E). The size andnumber of GFAP-positive astrocytes were significantly(See figure on next page.)
Fig. 1 Assessment of the degree of brain damage and behavioral changes in HI mice. A Flow chart of HI modeling and experimental design. Miceare ligated with the right common carotid artery at P7 postnatal, then hypoxia for 1 h (7% O2), tissue harvest is performed at P8, P14 and P60,
and behavioral tests are conducted at P60. B Representative images of TTC-stained coronal brain sections from sham and HI mice 24 h post-HI. The
red areas indicate the reaction of TTC reagent with mitochondrial respiratory enzymes in living tissues, while the white areas represent infarcted
tissue. Notably, large cerebral infarctions are observed in the HI group. C Mean and standard error of the infarct volume (two-tailed unpaired
Student’s t-test, n = 6 mice/group). D Representative trace track of mice in Y-maze test. Start arm(S), Old arm(O), New arm(N). E Time to explore
within the new arms (two-tailed unpaired Student’s t-test, n = 8 mice/group). F Representative trace track of mice in the novel object recognition
test. G Discrimination index (two-tailed unpaired Student’s t-test, n = 8 mice/group). H, I Total distance and center exploration time in the open field
experiment (two-tailed unpaired Student’s t-test, n = 8 mice/group). J Immobile time in the forced swimming (two-tailed unpaired Student’s t-test,n = 8 mice/group). K Time in the open arm of the elevated O-maze (two-tailed unpaired Student’s t-test, n = 8 mice/group)

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increased in HI group from P8-P14. HI mice had formed
glial scarring at P60 (Fig. 2F, G).
Differential metabolites and their functional analysis
on the sham and HI mice

We analyzed the HI related changes of metabolites in
the hippocampus. As depicted in Fig. 3A, The OPLSDA score plot demonstrates a clear separation between
the sham group (orange spots) and the HI group (green
spots), suggesting a metabolic distinction between thetwo conditions. Using VIP value > 1 and p-value < 0.05 as
criteria, we identified 48 downregulated and 119 upregulated differential metabolites (DEMs), including pyruvate, lactic acid, succinate, and citric acid (Fig. 3B). Morespecific information on differential metabolites can beseen in Supplementary Fig. S1. The hierarchical cluster ofthe DEMs (Fig. 3C) clearly shows metabolite differences
between the sham and HI group. KEGG analysis also
indicated that the metabolic pathways associated withalanine, aspartate, glutamate and pyruvate were enriched(Fig. 3D).
Functional analysis of differentially expressed genes
between sham and HI miceRNA-seq analysis revealed 60 downregulated and
147 upregulated differentially expressed genes(DEGs), using criteria of |log2 fold change|> 1 and
P < 0.05(Fig. 4A). The differences of gene expression
between the sham and HI groups were distinct as
depicted by the hierarchical cluster analysis (Fig. 4B).
Then, we conducted GO and KEGG analyses to investigate the functional implications of the DEGs. GO
analysis showed that the DEGs were associated withoxidative stress and inflammation, including woundhealing, activation of immune response, regulation of
IL-1, IL-6 and inflammatory et. al. (Fig. 4C). KEGG
enrichment analysis revealed that these DEGs wereclosely related signaling pathways of MAPK, NF-κB,
TNF, HIF-1α and apoptosis et. al. (Fig. 4D). At thesame time, anti-inflammatory and antioxidant pathways, including cellular response to extracellular

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stimulus, negative regulation of leukocyte activationand oxidative stress-induced intrinsic apoptotic arealso enriched. Figure 4E, F display the expression levelsof DEGs associated with oxidative stress/inflammation
and antioxidant /anti-inflammatory processes in shamand HI mice. These results suggest that during theacute phase of HI, both oxidative stress/inflammatory
responses and anti-inflammatory/antioxidant mechanisms were activated.
Protective effects of 4OI on HI mice

Based on the results of omics sequencing, we presumed that 4OI, an anti-oxidant and anti-inflammatory agent, may play a protective role in HI mice. We
injected 4OI intraperitoneally with 10, 25 and 50 mg/
kg in HI mice after HI. The mean infarct volume significantly decreased from 59.39% to 31.88% in the HI + 4OI(10 mg/kg) group compared to the HI group. A further
reduction to 14.92% was seen with 25 mg/kg 4OI. Interestingly, there was no significant difference between
the group receiving 50 mg/kg 4OI (average infarct area
14.55%) and the 25 mg/kg 4OI group (Fig. 5A, B). As
a result, we chose to use 25 mg/kg 4OI for the subsequent experiments. We performed negative geotaxis
and righting reflex tests to evaluate the short-term
neurological function at 24 h post HI. HI mice exhibited a prolonged latency in both negative geotaxis andrighting reflex tests. Treatment of 4OI significantly
reduced the latency in HI mice (Fig. 5C, D). NeuN
staining showed that 4OI treatment partially reduced
neuronal death, microglia and astrogliosis activationin hippocampal CA1 of HI mice (Fig. 5E, F). In addition, we observed 4OI can also effectively reduce rightbrain atrophy in adult HI mice (Fig. 5G, H). Moreover,the behavior performance of HI mice in the Y maze andnovel object recognition test was significantly improvedby 4OI treatment (Fig. 5I, J). Taken together, our results
indicate that 4OI treatment effectively alleviate pathological damage in hippocampus and cognitive dysfunction in HI mice.

(See figure on next page.)
Fig. 2 Changes of hippocampal cell numbers in sham and HI mice. A Representative gross morphology of mice Brains at P8, P14, and P60 (Left),
and the morphological structure of hippocampus in HI mice at P8, P14, and P60 (Right). Blue: DAPI. A noticeable atrophy of the hippocampus
on the ligated side in HI mice can be observed at P60. B Neun immunofluorescence staining of hippocampal tissue sections, showing
a significant reduction of Neun-positive neurons in HI mice. Red: Neun, Blue: DAPI, scale bar = 100 μm. C Mean and standard error of the numberof Neun-positive neurons (two-tailed unpaired Student’s t-test, n = 5 mice/group). D Iba1 immunofluorescence staining, demonstrating a marked
elevation in Iba1-positive microglia in HI Mice. Green: Iba1, Blue: DAPI, white arrows: activated microglial cells. E The number of Iba1-positive
microglia (two-tailed unpaired Student’s t-test, n = 5 mice/group). F GFAP immunofluorescence staining, indicating a pronounced increasein GFAP-positive astrocytes in HI Mice. Red: GFAP, Blue: DAPI, white arrows: activated astrocytes. G The number of GFAP-positive astrocytes(two-tailed unpaired Student’s t-test, n = 5 mice/group). Cell counting was limited to the hippocampal region, excluding areas such as the cortex.
This applies to all quantifications mentioned in panels C, E, and G

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Yang et al. Journal of Neuroinflammation(2024) 21:132

4OI upregulates Nrf‑2 expression and exerts
anti‑inflammatory and antioxidant effects.

Some studies showed that 4OI can promote the dissociation of Nrf2 and Keap1, Nrf2 nuclear translocation andNrf2-dependent gene expression [27, 40]. Our immunofluorescence staining for NeuN (Fig. 6A), Iba1(Fig. 6B)
and GFAP (Fig. 6C) indicated a clear co-localization ofastrocytes with Nrf2 in the hippocampus of HI mice.
After treatment with 4OI, reactive astrocytes exhibiteda decrease in cell body size, indicative of a reversion toa quiescent state, and there was a significant increase in
the number of ­Nrf2+GFAP+ cells. These results suggestthat 4OI not only suppresses the activation of astrocytes but also enhances their Nrf2 expression (Fig. 6D).
Figure 6E illustrates the colocalization of Nrf2 with thenucleus in astrocytes, showing that the HI group had lessNrf2 within nucleus, while 4OI treatment significantly
increased the number of Nrf2 in the nucleus (Fig. 6F).
Therefore, 4OI has the effect of increasing the level ofNrf2 and promoting its nuclear translocation. RT-PCR
results revealed a substantial elevation in the expression
levels of IL-1β, IL-6, C3, Ctla2a and Ifi211 in the HI groupcompared to the sham group. Notably, this increase was
effectively mitigated by the treatment of 4OI (Fig. 6G, H).
In addition, the Nfe2l2, HO-1 and Atf3 expression levels
were elevated in the HI group and further increased with
4OI treatment (Fig. 6I, J). These results suggest that 4OI
may inhibit the production of pro-inflammatory factors
by promoting the expression of antioxidant enzymes.
The effects of 4OI on Nrf2 in astrocytes and microglia

Previous studies on the inflammatory models induced
by LPS have shown that 4OI primarily exerts its antiinflammatory effects on microglial/macrophage cells[31, 41]. Our in vivo studies on the HI model also confirmed the anti-inflammatory and antioxidative effects
of 4OI. However, our results suggest that 4OI may exert
its effects by promoting the nuclear translocation of Nrf2
in astrocytes, without significantly affecting Nrf2 expression and nuclear translocation in microglial cells. To further confirm the cellular targets of 4OI, we established an
in vitro OGD/R model to examine the effects of 4OI on

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Nrf2 expression and nuclear translocation in astrocytes(CB-D1A) and microglia (BV2).
Nrf2 was significantly expressed in the cytoplasmof CB-D1A cells in both control and OGD/R groups,
with lesser expression in the nucleus. However, in the
OGD/R + 4OI group, a noticeable increase in nuclear
Nrf2 was observed. In BV2 cells, Nrf2 expression
was limited to the cytoplasm in control, OGD/R and
OGD/R + 4OI groups. Moreover, Western blot results
further confirmed that 4OI increased the expressionof Nrf2 in astrocytes, while it had no significant effecton Nrf2 expression in BV2 cells. These findings suggestthat 4OI primarily enhances Nrf2 expression and nuclear
translocation in astrocytes (Fig. 7).
Silencing the Nrf2 of astrocytes can attenuate
the anti‑inflammatory and antioxidant effects of 4OI

To validate specific 4OI protection, we used the Nrf2
siRNA constructs to reduce the expression of Nrf2 in
C8-D1A cells, and then exanimated the effect of 4OI
in rectifying the abnormal expression of inflammatory
factors and antioxidant enzymes induced by OGD/R.
Western blot analysis confirmed successful transfectionof both Nrf2-1 siRNA and Nrf2-2 siRNA constructs,
effectively reducing Nrf2 protein levels in C8-D1A cells(Fig. 8A). As both si-Nrf2-1 and si-Nrf2-2 showed similar efficacy in suppressing Nrf2 protein, Nrf2-1 siRNAwas chosen for subsequent experiments. Immunofluorescence staining further validated the reduction in Nrf2
fluorescence intensity in C8-D1A cells transfected with
Nrf2-1 siRNA plasmid (Fig. 8B). Subsequent analysis
revealed that 4OI treatment significantly upregulated
Nrf2 and HO-1 expression while downregulating IL-1β
levels after OGD/R in normal C8-D1A cells. However,these effects were attenuated in Nrf2-1 siRNA-transfected C8-D1A cells, indicating that 4OI mediates its
anti-inflammatory and antioxidative effects through Nrf2
in astrocytes.

Discussion
In this study, we firstly employed an approach combining untargeted metabolomics with transcriptomics toscreen for changes in metabolites and gene expression(See figure on next page.)
Fig.3 Metabolites and pathways associated with HI. A OPLS-DA score plot of sham (green) and HI group (orange). B Volcanic diagram of DEMs.
Blue denotes down-regulated metabolites(VIP ≥ 1, P < 0.05, FC > 0), red denotes up-regulated metabolites(VIP ≥ 1, P < 0.05, FC < 0) and grey denotes
insignificant metabolites. C Hierarchical cluster of the DEMs. The left vertical axis shows clusters of DEMs, while the above horizontal axis showsclusters of samples. Red represents high expression and green represents low expression. D The bubble chart of KEGG enrichment based on DEMs.
The abscissa represents the rich factor corresponding to each pathway, the ordinate represents the pathway name, and the color of the pointis the p-value. The redder the point is, the more significant the enrichment is. The size of the dot represents the number of enriched differential
metabolites

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in a mouse model of acute HI. The results revealed a significant accumulation of lactate and succinate, as wellas elevated expression of inflammatory genes related to
the IFN and NF-κB pathways. Additionally, we observed
increased expression of antioxidant stress genes, such as
Atf3, Nfe2l2, and Hmox-1. Furthermore, intraperitoneal
injection of 4OI in HI mice reduced the infarct area, suppressed the proliferation of microglia and astrocytes, andmitigated neuronal death, resulting in improved cognitive function in adult HI mice. The neuroprotective
effects of 4OI were potentially mediated through upregulation of Nrf2 levels in astrocytes, inhibition of IL-1β andIL-6 production, and increased expression of HO-1 andAtf3. Our findings contribute to a better understandingof the complex pathophysiological mechanisms underlying HIE and provide insights for the development of targeted therapeutic interventions.
Clinical researchers have found that HIE patients often
exhibit significant cognitive impairments in adulthood,including difficulties in learning, memory issues, andattention deficits [42–45]. The damage to hippocampal
neurons caused by ischemia and hypoxia is likely a key
pathological process in this context. In our study, weobserved pronounced neuronal death in the CA1 regionof HI mice at P8, with neuronal deaths in the CA1 region
further increasing over time without intervention. Concurrently, there was a significant proliferation and activation of microglia and astrocytes in the CA1 area. By P60,
the ipsilateral hippocampal region in the HI mice showed
atrophy and astroglial scar. Neurons in the hippocampal CA1 region have been proven to play a crucial rolein cognition, spatial learning, and memory processes [46,47]. Related studies have already demonstrated memory
impairment in the HI model [48, 49]. Similarly, in ourstudy, adult HI mice exhibited a marked reduction in
exploration time of novel arms and objects in the Y-mazeand novel object recognition tests. These findings further
confirm the central role of hippocampal neuronal damage in cognitive dysfunction caused by HIE.
Previous studies indicated that the initial pathological process (0–6 h) of HIE is characterized by an energy
deficit. Hypoxia compels cells to transition from the tricarboxylic acid (TCA) cycle to anaerobic glycolysis as aPage 13 of 21

compensatory energy source, leading to an increased
production of pyruvate and lactate. High levels of lactate
and pyruvate can increase the production of reactive oxygen species (ROS) and inflammatory mediators, damage
the blood–brain barrier (BBB), and lead to edema. Thesechanges eventually result in sustained neuronal damage
within 6–48 h post-injury [13, 21, 50, 51]. Consistent
with this, we also observed elevated levels of pyruvateand lactate in the hippocampal tissue of acute HI mice,indicating a greater reliance on glycolysis by the cells.
Our results of high-throughput transcriptomics revealed
upregulated expression of genes related to oxidative
stress and inflammation. Further analyses confirmed thatthese gene expression changes are associated with biological processes related to IFN and IL-1β/6, and the KEGG
pathways of NF-κB, apoptosis, HIF-1α. This suggests thatoxidative stress and inflammatory responses may be theprimary factors contributing to neuronal damage in the
hippocampus.
On the other hand, concomitant with the occurrence ofoxidative stress and inflammatory responses, anti-inflammatory and antioxidant mechanisms are also triggered.
One of a key molecule of endogenous antioxidant andanti-inflammatory responses is Nrf2. Under oxidative
stress, Nrf2 dissociates from Kelch-like ECH-associatedprotein 1 (Keap1) and translocates to the nucleus, where
it binds to antioxidant response elements (AREs) and
upregulates the expression of various antioxidant genes,
notably HO-1 [52]. Additionally, Nrf2 inhibits the expression of pro-inflammatory cytokines, such as IL-1β and
IL-6, by modulating the Atf3/IκBζ pathway [53–56]. Our
study also found that the expressions of Nfe2l2, Hmox1,
and Atf3 were elevated in the hippocampus of HI mice,suggesting that the mechanisms of anti-inflammatory
and antioxidant are triggered during the early stagesof HI. These results imply that enhancing the antioxidant and anti-inflammatory response mediated by Nrf2
may play a neuro-protective role in HI. Furthermore,
our immunofluorescence staining results revealed thatthe expression level of Nrf2 significantly increased in HI
mice, and Nrf2 predominantly co-localizes with astrocytes, indicating that HI predominantly triggers Nrf2
expression in astrocytes. Previous study has also reported(See figure on next page.)
Fig.4 Enrichment analysis of DEGs associated with HI. A Volcano map shows DEGs between sham vs HI group. Red: upregulated DEGs (P < 0.05,
log2FC > 1); blue: downregulated DEGs (P < 0.05, log2FC < 1). B Hierarchical cluster analysis. Different columns in the figure represent different
samples, and different rows represent different genes. The colors from blue to yellow indicate gene expression from low to high, respectively. C, D
The bubble chart of GO and KEGG enrichment based on DEGs. The ordinate represents enriched GO functional and KEGG pathway terms of these
DEGs, and the abscissa represents the –log 10 (P-values) of the significantly enriched terms. Counts represent the number of DEGs enriched in each
GO functional or KEGG pathway term. E Heatmap showing the 22 DEGs associated with IL-1, IL-6, immune response and inflammatory response. F
Heatmap showing the 17 DEGs associated with negative regulation of apoptotic, leukocyte activation and response to hypoxia

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Fig. 5 Effect of 4OI treatment on brain histopathological changes and cognitive dysfunction in HI mice. A Representative TTC stained coronalbrain sections from Sham, HI and HI + 4OI groups 24 h post-HI, it was found that ≥ 25 mg/kg 4OI could significantly reduce the cerebral infarctionfoci of HI mice. B Infarct volume (one-way ANOVA with Tukey’s multiple-comparison test, n = 4 mice/group). C, D The latency time of righting
reflex and geotaxis reflex. Compared with the Sham group, the latency time of HI mice was significantly prolonged, and significantly shortened
after 4OI treatment (one-way ANOVA with Tukey’s multiple-comparison test, n = 8 mice/group). E, F Neun, Iba1, and GFAP immunofluorescencestaining of hippocampal tissue sections. Counts of Neun-positive neurons, Iba1-positive microglia and GFAP-positive astrocytes. The 4OI
treatment is observed to effectively mitigate the reduction of hippocampal neurons and the proliferation of microglia and astrocytes in HI mice(one-way ANOVA with Tukey’s multiple-comparison test, n = 5 mice/group). scale bar = 100 μm. Cell counting was limited to the hippocampal
region, excluding areas such as the cortex. G, H Comparison of brain morphology and weight in different groups at P60. The right-to-left
cerebrum (ipsilateral/contralateral) weight ratio was smaller in HI group, suggesting a significant tissue loss in the ipsilateral hemisphere, which
was significantly relieved after 4OI treatment (one-way ANOVA with Tukey’s multiple-comparison test, n = 4 mice/group). I, L 4OI treatment
notably increased exploration time in the Y-maze and discrimination index in the object recognition test (one-way ANOVA with Tukey’s
multiple-comparison test, n = 8 mice/group)

Yang et al. Journal of Neuroinflammation(2024) 21:132

that multiple Nrf2-targeted antioxidant and detoxification genes are preferentially expressed in glial cells [57,58]. Indeed, the protective role of Nrf2 in astrocytes has
been shown in mouse models of ALS, Parkinson’s disease,cerebral hypoperfusion and Alzheimer’s disease with theastrocyte-specific Nrf2 transgene [59]. Thus, astrocytes
may be the primary cell type where Nrf2 exerts its antioxidative and anti-inflammatory effects.
Recent studies have shown that 4OI, a derivative of
itaconate with high cell permeability, is one of a valid
Nrf2 activator. It facilitates the dissociation of Keap1
and Nrf2, reducing the ubiquitination of Nrf2 and promoting its nuclear translocation to regulate the expression of downstream genes related to antioxidant andanti-inflammatory responses [60]. In vitro study showsthat 4OI can protect SH-SY5Y cells and epigeneticallyde-repressed primary neurons from H­ 2O2 [61]. In vivo
experiments have also confirmed that 4OI can alleviate β2M-induced cognitive dysfunction and hippocampal neurogenesis impairment [62]. For the first time,
we examined the effects of 4OI as a therapeutic agent
for HIE. Our results show that 4OI effectively reduces
the infarct area in HI mice, inhibits the activation andproliferation of microglia and astrocytes, and decreases
neuronal apoptosis. Furthermore, 4OI significantly
alleviates brain atrophy in adult HI mice and mitigates
their cognitive dysfunction. In HI mice, the expression
level of Nrf2 in astrocytes mainly increased in the cytoplasm. However, 4OI treatment increased the expression level of Nrf2 in both cytoplasm and nucleus. In
addition, our in vitro experiments have demonstrated
that 4OI can promote Nrf2 expression and nuclear
transfer in astrocytes. Simultaneously, the levels ofNfel2l2, HO-1 and Atf3 are significantly increased in
the 4OI treatment group, and the levels of IL-1β, IL-6,
C3, Ctla2a and Ifi211 are significantly decreased. In

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addition, after knocking down the expression of Nrf2
in astrocytes cultured in vitro with the Nrf2 siRNA
plasmid, we found that the anti-inflammatory and antioxidant effect of 4OI against OGD/R was significantly
diminished, suggesting that 4OI exerts anti-inflammatory and antioxidant effects through Nrf2 in astrocytes.
There are also some shortcomings in this study. First,
the sample size of the included studies in this experiment
was small, which did not fully validate the safety and efficacy of 4OI as a therapeutic drug. Secondly, this study
did not involve clinical data and could not confirm theactual therapeutic effect of 4OI on HIE patients. Finally,
our in vivo experiments used Bulk-RNA sequencing andnon-targeted metabolome sequencing, and the results
did not determine the specific cell type. Although our
results from in vivo immunofluorescence staining andin vitro cell culture experiments suggest that 4OI canregulate Nrf2 expression and function in astrocytes, itcannot be ruled out that 4OI also plays a similar role inother cell types.
In conclusion, we found that in the acute phase of HI,
there is an accumulation of pyruvate and lactate in the
hippocampal tissue, accompanied by oxidative stress andpro-inflammatory, as well as increased expression of antioxidative stress and anti-inflammatory genes. We also
confirmed that 4OI exerts neuroprotective effects against
HI through the Nrf2 pathway in astrocytes. It is important to note that our experimental observations havefocused on the early stages of HI, and it remains unclear
whether Nrf2 plays a similar role in other cell types in
the late HI stages. This issue deserves further study. Nevertheless, our research contributes to a more comprehensive understanding the complex pathophysiology of
HIE and provides new insights for targeted therapeuticinterventions.

(See figure on next page.)
Fig. 6 Effects of 4OI on Nrf2 expression, inflammatory cytokines and antioxidant enzymes. A-C Immunofluorescence staining of Nrf2 with NeuN,
Iba1, and GFAP in the mouse hippocampus. These images demonstrate a higher colocalization of Nrf2 with GFAP and a comparatively lower
colocalization with NeuN and Iba1 (n = 5 mice/group). Cell counting was limited to the hippocampal region, excluding areas such as the cortex.
D Statistical graph of the count of Nrf2-positive cells (one-way ANOVA with Tukey’s multiple-comparison test, n = 4 mice/group). The proportionof ­Nrf2+GFAP+ cells is higher in the HI group compared to the Sham group, and even higher in the HI + 4OI group. The proportion of ­Nrf2+
cells is lower in both ­NeuN+ and ­Iba1+ cells across all groups. E Representative plot illustrating the spatial relationship between Nrf2 and DAPIin ­GFAP+ cells. Lesser colocalization of Nrf2 with DAPI is observed in both the Sham and HI groups, with an increased colocalization in the HI + 4OI
group. scale bar = 100 μm. F Statistical plot of the count of ­GFAP+ cells with Nrf2 expression in the nucleus (one-way ANOVA with Tukey’s
multiple-comparison test, n = 4 mice/group). G Quantitative analysis of IL-1β, IL-6, C3, Ctla2a, Ifi211, Nfel2l2, ATF3, HO-1 and Atf3 mRNA levelsin the hippocampus of mice 24 h post-HI using RT-PCR. The 4OI treatment significantly reduced the expression levels of IL-1β, IL-6, C3, Ctla2aand Ifi211while it enhanced the expression of Nfel2l2, HO-1 and ATF3 in the HI mice (two-way ANOVA with Tukey’s multiple-comparison test, n = 3
mice/group)

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Fig. 7 The effect of 4OI on Nrf2 expression levels in BV2 and C8-D1A cells. A Immunofluorescence staining of Nrf2 in C8-D1A cells. It can beobserved that the expression of Nrf2 in the cytoplasm of the control group and the OGD/R group was more than that in the nucleus, while in the
OGD/R + 4OI group, there is no significant difference in Nrf2 expression between the cytoplasm and nucleus. scale bar = 100 μm. B, C Representative
picture of Western blot data from C8-D1A astrocytes. The quantitative protein analysis graph showed that 4OI significantly increases the expression
level of Nrf2 (one-way ANOVA with Tukey’s multiple-comparison test, n = 3 /group). C Immunofluorescence staining schematic of Nrf2 in BV2 cells.
In all three groups, Nrf2 expression is higher in the cytoplasm than in the nucleus. scale bar = 100 μm. D, E Representative picture of Western blot
data from BV2 microglia. The quantitative protein analysis graph showed no significant difference in Nrf2 expression levels among the three groups(one-way ANOVA with Tukey’s multiple-comparison test, n = 3 /group)

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Fig. 8 Effect of Nrf2 on HO-1 and IL-1β expression in silencing C8-D1A cells. A Western blot data and protein quantification charts of Nrf2
protein in C8-D1A cells in the Control, si-Negative and si-Nrf2 groups showed that the expression of Nrf2 protein in the si-Nrf2-1 and si-Nrf2-2
groups was significantly reduced (one-way ANOVA with Tukey’s multiple-comparison test, n = 3 /group). B Immunofluorescence staining of Nrf2
expression in C8-D1A cells after Nrf2 siRNA transfection, showing that the immunofluorescence intensity of Nrf2 in the si-Nrf2-1 group decreased.
scale bar = 100 μm. C Western blot data and protein quantification charts of Nrf2, IL-1β and HO-1 in C8-D1A cells with OGD/R, OGD/R + 4OI
and OGD/R + 4OI + si-Nrf2-1 groups (two-way ANOVA with Tukey’s multiple-comparison test, n = 3 /group). In the si-Nrf2-1 + OGD/R + 4OI group,transfection of Nrf2 siRNA inhibited the effect of 4OI on the promotion of Nrf2 and HO-1 expression and the inhibition of IL-1β expression

Abbreviations
HIE	Hypoxic ischemic encephalopathyHI	Hypoxic ischemic
4OI	4-Octyl itaconate
Nrf2	Nuclear factor erythroid-2-related factor
HO-1	Hmox1
TTC​	2,3,5-Triphenyltetrazolium chloride

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03121-8.
Supplementary Material 1 (PDF 45 KB)

Author contributions
YPY performed part of the experiments and wrote the initial draft of themanuscript; YL carried out the data analysis; WYY, ML and XYY performed partof the experiments and revised the manuscript; LQ and JCZ were primarily
responsible for experiment design and manuscript preparation.
Funding
This work was supported by the following grants: the projects for Department of Science and Technology of Liaoning Province (2020JH2/10100014,
2021JH1/10400049 to LQ); Xingliao Talent Plan of Liaoning Province, China(XLYC2002094 to LQ).
Availability of data and materials
Data supporting the findings of this study are available upon reasonable
request to the corresponding author.

Yang et al. Journal of Neuroinflammation(2024) 21:132

Declarations
Ethics approval and consent to participate
All animal experiments were approved by the Institutional Animal Care and
Use Committee of China Medical University (KYDWCS2023534) and conducted in accordance with the ethical standards.
Consent for publication
All the co-authors and participants have given their consent for publication in
Journal of Neuroinflammation.
Competing interests
The authors declare that they have no competing interest.
Author details
1
Department of Anesthesiology, The Shengjing Hospital of China Medical
University, Shenyang, Liaoning, China. 2 Department of Physiology, China
Medical University, Shenyang, Liaoning, China. 3 Department of Anesthesiology, Shenzhen Cancer Hospital, Shenzhen, China.
Received: 26 December 2023 Accepted: 1 May 2024

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