Journal of Neuroinflammation

Guo et al. Journal of Neuroinflammation(2024) 21:131
https://doi.org/10.1186/s12974-024-03111-w

Open Access

RESEARCH

Therapeutic effects of orexin-A in sepsisassociated encephalopathy in mice
Jing Guo1, Zhuo Kong3, Sha Yang1, Jingjing Da4, Liangzhao Chu5, Guoqiang Han3, Jian Liu3*, Ying Tan3* andJiqin Zhang2*
Abstract
Background Sepsis-associated encephalopathy (SAE) causes acute and long-term cognitive deficits. However,information on the prevention and treatment of cognitive dysfunction after sepsis is limited. The neuropeptide
orexin-A (OXA) has been shown to play a protective role against neurological diseases by modulating the
inflammatory response through the activation of OXR1 and OXR2 receptors. However, the role of OXA in mediating
the neuroprotective effects of SAE has not yet been reported.
Methods A mouse model of SAE was induced using cecal ligation perforation (CLP) and treated via intranasal
administration of exogenous OXA after surgery. Mouse survival, in addition to cognitive and anxiety behaviors,
were assessed. Changes in neurons, cerebral edema, blood-brain barrier (BBB) permeability, and brain ultrastructure
were monitored. Levels of pro-inflammatory factors (IL-1β, TNF-α) and microglial activation were also measured. The
underlying molecular mechanisms were investigated by proteomics analysis and western blotting.
Results Intranasal OXA treatment reduced mortality, ameliorated cognitive and emotional deficits, and attenuatedcerebral edema, BBB disruption, and ultrastructural brain damage in mice. In addition, OXA significantly reduced
the expression of the pro-inflammatory factors IL-1β and TNF-α, and inhibited microglial activation. In addition, OXA
downregulated the expression of the Rras and RAS proteins, and reduced the phosphorylation of P-38 and JNK, thus
inhibiting activation of the MAPK pathway. JNJ-10,397,049 (an OXR2 blocker) reversed the effect of OXA, whereas
SB-334,867 (an OXR1 blocker) did not.
Conclusion This study demonstrated that the intranasal administration of moderate amounts of OXA protects the
BBB and inhibits the activation of the OXR2/RAS/MAPK pathway to attenuate the outcome of SAE, suggesting thatOXA may be a promising therapeutic approach for the management of SAE.
Keywords Orexin-A, Sepsis-associated encephalopathy, Inflammation, Cognitive dysfunction, Orexin receptors*Correspondence:
Jian Liuliujian@gz5055.com
Ying Tantanying@gz5055.com
Jiqin Zhang
zhangjiqin@gz5055.com
1
GuiZhou University Medical College, Guiyang 550025, Guizhou Province,
China

2

Department of Anesthesiology, Guizhou Provincial People’s Hospital,
Guiyang, China
3
Department of Neurosurgery, Guizhou Provincial People’s Hospital,
Guiyang, China
4
Department of Nephrology, Guizhou Provincial People’s Hospital,
Guiyang, China
5
Department of Neurosurgery, the Affiliated Hospital of Guizhou Medical
University, Guiyang, China© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Guo et al. Journal of Neuroinflammation(2024) 21:131

Introduction
Sepsis is a systemic inflammatory response caused
by infection or suspected infection, and is the leading
cause of morbidity and mortality in intensive care units.
Sepsis-associated encephalopathy (SAE) is defined as
widespread brain dysfunction resulting from a systemic
inflammatory response, and is characterized by disruption of the sleep-wake cycle, confusion, cognitive dysfunction, and coma [1]. SAE significantly impacts patient
prognosis [2, 3], and studies have shown that sepsis survivors remain at risk of long-term sequelae, such as cognitive impairment and depression [4]. Clinical management
primarily focuses on treating symptoms, and rapid fluid
resuscitation is a key therapeutic strategy. However,there is a significant risk of complications including
metabolic acidosis and hyperkalemia [5]. In recent years,
several neuroprotective agents have been reported for
SAE, including ginsenosides [6] and glucocorticoids [7].
However, information regarding the treatment of cognitive impairment in sepsis is sparse [8]. Therefore, developing effective treatment strategies for this condition isimportant.
The blood-brain barrier (BBB) comprises endothelial
cells of the cerebral capillaries, pericytes, astrocytes, andtight junctions within the basement membrane [9], andserves as the brain’s defense mechanism against foreign
pathogens and toxins in the blood [10]. In sepsis, the barrier function of the BBB is reduced or absent, allowing alarge number of inflammatory factors and cells from theperipheral blood enter the brain tissue through the damaged or collapsed BBB, leading to brain damage [11]. As
such, mitigation of BBB damage plays an important rolein improving the prognosis of SAE.
The primary mechanism underlying sepsis is an uncontrolled inflammatory response. As the body’s inflammatory cascade response develops, a significant number of
inflammatory cells accumulate at the site of injury, causing functional damage to various organs, including thebrain tissue [4]. Blocking microglial activation and reducing the production of toxic factors after activation may
facilitate SAE treatment [12, 13]. As such, the rational
modulation of inflammation is critical for optimizing theoutcomes of SAE [14–16], while effective drugs to reducethe incidence and severity of infectious brain injury arenecessary.
Recent studies have shown that the nervous system
directly influences the course of SAE via the productionof neuropeptides [17]. Orexin is an excitatory neuropeptide synthesized and secreted by the hypothalamus.
Orexin is classified into two types: orexin A (OXA) andorexin B (OXB). OXA and OXB bind to the G proteincoupled receptors orexin-1 receptor (OXR1) and orexin-2
receptor (OXR2), respectively, to regulate several important physiological activities in organisms [18]. OXA has

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the same affinity for OXR1 and OXR2, whereas OXB
preferentially binds to OXR2 [19]. Because OXB is rapidly
degraded in the circulation following peripheral administration, the intact form of OXB cannot be detected in thebrain, while OXA can cross the BBB mainly by simple diffusion [20]. Intranasal administration has been validated
as a delivery strategy to allow rapidly delivery of OXA to
the CNS with minimal systemic exposure [21]. Given thisinformation, in the present study, we focused on OXA
administered intranasally. A large number of previous
studies have shown that an imbalance in OXA is associated with Parkinson’s disease [22], Alzheimer’s disease[23], and other neurological disorders. Animal studies
have further shown that OXA reduces neuroinflammation [24, 25], inhibits the endothelial cell inflammationinduced by oxidized low-density lipoproteins in endothelial cells [26], and inhibits neuronal apoptosis [27]. Thus,
OXA may be a potential therapeutic drug for central
nervous system cognitive disorders. However, no studies have yet confirmed the therapeutic effects and exact
mechanism of action of OXA on SAE.
In the present study, we found that OXA had a therapeutic effect on cecal ligation perforation (CLP)-induced
pathological injury and cognitive dysfunction in SAE,
and elucidated that the mechanism may be related to
BBB protection and the inhibition of OXR2/RAS/MAPK
pathway activation. Our findings not only provide neuropeptide candidates for the treatment of SAE, but also
provide important insights into the pathological mechanisms and potential drug targets.

Materials and methods
Animals

Male C57BL/6J mice (6–8 weeks old, 20–25 g) were purchased from Tengxin Biotech (Chongqing, China). All
mice were maintained at 22 ± 2 °C under a 12-h light/darkcycle with water and food ad libitum. This animal study
was approved by the Guizhou Provincial Institutional
Animal Care and Use Committee of the Provincial People’s Hospital (Guizhou, China).
Preparation of CLP-induced septic mouse model

The modelling method was based on previous protocols[28, 29]. In brief, mice were anesthetized by sevoflurane
inhalation. After depilation, the abdomen was sterilized and a longitudinal incision of approximately 1.0 cm
was made along the midline. The cecum was carefully
removed with sterile forceps, ligated with sterile 5 − 0silk, and perforated at the blind end with a 22G injectionneedle to avoid damage to the mesentery and cecal vessels. After a small amount of fecal matter was expelled
by gentle squeezing of the cecum, it was returned to theabdominal cavity. The Sham group underwent the sameprocedure but without ligation and perforation. Finally,

Guo et al. Journal of Neuroinflammation(2024) 21:131

the abdominal incision was closed in layers, and all mice
were injected intraperitoneally with saline (5 ml/100 g) toreplenish the body fluids lost during surgery.
AdministrationOXA (HY-106,224, MedChemExpress) was dissolvedin saline and the OXA drug solution was administeredintranasally. OXA was administered at three dose levelsof 50 µg/kg (low dose), 250 µg/kg (medium dose), and500 µg/kg (high dose) [30], 30 min after CLP induction[31], in a volume of 2.5 µl per nostril (5.0 µl per mouse).
In the OXA optimal dose experiment, mice were intranasally administered OXA 250 µg/kg once daily for 7 days.
SB-334,867 (HY-10,895, MedChemExpress) [32, 33]
and JNJ-10,397,049 (HY-108,906, MedChemExpress)[34], which are selective antagonists for OXR1 andOXR2, respectively, were dissolved in 5% DMSO to forma drug solution (10 mg/ml). Thirty minutes before CLP
induction, 100 µL of the solution was injected intraperitoneally (i.p.), while the control group received the same
volume (100 µl) of 5% DMSO.
Open field test (OFT)The OFT is a classic behavioral test used to assess spontaneous activity, anxiety, and exploratory behavior inanimals. This test was performed 1 and 7 days after surgery. The experimental apparatus comprised an opaque
white cubic box measuring 50 × 50 × 40 cm. Each mouse
was placed in a box with its back on one side of the box
wall, and allowed to explore freely for 5 min. The total
distance travelled and time spent were recorded using an
automatic video tracking system (RWD Life Science Co.
Ltd., China). The apparatus was wiped with 75% alcohol
to remove odors.
In the OFT, the total distance travelled and the timespent in the central area were assessed.
Y-maze test (Y-maze)

The Y-maze test is primarily used to test discriminative
learning and working memory in animals. It consists ofthree white opaque arms, each measuring 30 × 5 × 20 cm(RWD Life Science Co., Ltd., China). In our experiments,
one arm was chosen as the “start arm,” another as the“novel arm” and the third as the “other arm.” The animal
was first placed in the “start arm” with its back to thecenter, after which the “novel arm” was closed. The animals were allowed to explore freely between the arms for
15 min during the training session. One hour later, theanimal was placed in the “start arm” and then returned tothe center. The “novel arm” was activated and the animals
were allowed five minutes to explore freely. The timespent by the mice on each arm and the distance travelled by the animals on each arm were recorded. These
measurements were used as indices to assess spatial

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recognition and memory. The maze was cleaned with
75% ethanol solution before each test.
In the Y-maze test, exploration and memory skills were
quantified as the percentage of time spent exploring the
new arm: %time = (zone C activity time/test time) × 100.
We further assessed the percentage of distance travelledwithin the new arm of the activity as follows: %travel distance = (zone C distance/total distance) × 100.
Tail suspension test (TST)The TST was used to evaluate depression-related behavior. This test was carried out one and seven days after surgery, according to a previously established method [35].
In a dark and quiet room, the mice were taped approximately 1 cm from the tip of their tails and suspended
upside down with their heads approximately 30 cm above
the top of the table. The resting time of each mouse wasrecorded for 5 min.
In the TST, anxiety-like behavior was quantified as thetotal time the mice remained stationary for 5 min.
Determination of brain water content

The mice were euthanized after surgery. The brain was
immediately excised and the wet weight was determined
It was then dried at 100 °C for 24 h and weighed again to
determine the dry weight. The water content of brain tissue was then calculated. The formula for calculating thepercentage water content of brain tissue is: (wet weightof brain tissue - dry weight of brain tissue) / wet weight
× 100%.
BBB permeability
Assessment of Evans blue dye extravasation

A 2% Evans blue (EB) solution (Sigma-Aldrich) was dissolved in saline and injected into the caudal vein at a doseof 4 ml/kg. After 1 h, all the animals were euthanized
and transcardially perfused with 0.9% saline for 20 min
to remove the intravascular dye. The hippocampus was
subsequently isolated from the brain, homogenized with
0.5 ml of 60% trichloroacetic acid and incubated at 4 °C
for 30 min. After centrifugation (12,000 rpm for 15 min),absorbance was measured at 620 nm using an enzyme
marker (BioTek, USA).
Endogenous immunohistological detection of IgG
extravasation

Mice were anesthetized, cardiac perfusion was performed, and the brains were removed and fixed in 4%
paraformaldehyde (PFA) at 4 °C for over 24 h. Sectionsof 20 μm thickness were then taken using a frozen section machine (Leica, Japan). The cells were then treatedwith Alexa Fluor 488-conjugated goat anti-mouse IgG(A-32,723, Invitrogen) for 1 h. The fluorescence intensity

Guo et al. Journal of Neuroinflammation(2024) 21:131

of IgG was determined using a research slide scanner(Zeiss, LSM 980).
Immunofluorescence (IF) staining

Mouse brain tissue was cryosectioned and incubated with
10% goat serum (G1208, Servicebio). Slices were thenincubated with rabbit anti-Iba-1 (EPR16588, Abcam)
overnight at 4 °C, and the samples were subsequently
treated with an Alexa Fluor Plus 594-conjugated goatanti-rabbit fluorescent secondary antibody (A-11,062,
Invitrogen) for 1 h. Finally, fluorescence images were captured using a laser scanning confocal microscope (Zeiss,
LSM 980) and analyzed using ImageJ software.
Western blotting

Hippocampal tissue was homogenized and centrifuged, and the supernatant was extracted. Proteins wereextracted according to the manufacturer’s instructions
and quantified using a BCA protein assay kit (Well-Bio,
China). Equal amounts of protein (30 µg) were separated on 10% acrylamide gels, and transferred to 0.22 μm
PVDF membranes. After blocking, primary antibodies
diluted in the antibody diluent were added for incubation at 4 °C overnight and then washed. The following
primary antibodies were used: rabbit anti-Rras (ER60170,
HuaBio), rabbit anti-Orexin-A (BS15009R, Bioss), rabbit anti-Orexin receptor 2 (Ab183072, Abcam), rabbit
anti-Orexin receptor 1 (#59,242), rabbit anti-Ras (#3965),
rabbit anti-IL-1β (#31,202), rabbit anti-phospho-MAPK
family (#9926) and MAPK family (#9926) (Cell Signaling Technology), rabbit anti-TNF-α (A11534, Abclonal).
Horseradish peroxidase (HRP)-conjugated secondary
antibody (GB23303, Servicebio) was added, and the mixture was washed three times at room temperature. The
results were analyzed using the ImageJ software.
Observation of ultrastructural changes by transmission
electron microscopy

Seven days after model establishment, mice were anesthetized and perfused with glutaraldehyde fixative, andthe hippocampus was rapidly removed and immersed in
a 2% glutaraldehyde solution for more than 24 h. They
were then fixed in 4% glutaraldehyde for 2 h, rinsed thricewith PBS, and fixed in 1% osmium for 2 h. Tissues were
dehydrated using an acetone gradient, polymerized at
37 °C and 60 °C in a thermostat, and sectioned at a thickness of approximately 50 nm using an ultrathin sectioning machine. Finally, the cell ultrastructure was observedby transmission electron microscopy after ultrathin double staining with uranyl acetate and lead acid.
Nissl staining

Mouse brains were collected 1 day after surgery to assess
hippocampal damage. The hearts were perfused with 4%

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paraformaldehyde-PBS solution and fixed in 4% paraformaldehyde solution for 24 h, then paraffin-embedded
before taking 5 μm thick coronal sections. The sections
were placed in Nissl staining solution (G1036, Servicebio)
and neurons in the CA1 area of the hippocampus wereobserved under a light microscope (Olympus, Japan).
Quantitative proteomic analysis

Hippocampal tissues from the brains of mice in the sham,
CLP, and OXA optimal dose (250 µg/kg) groups wererapidly harvested 7 days after surgery and stored in liquid nitrogen. Proteomic identification was performed byHangzhou Jingjie Biotechnology Co. on nine mice (three
mice per group). Samples were collected for protein concentration using a BCA kit. Samples were then desalted
on a Strata X C18 solid-phase extraction column (Phenomenex) and dried under vacuum. Separation was performed using high-pH reversed-phase high-performanceliquid chromatography. Tryptophan peptides weretreated and applied to an NSI source before being subjected to tandem mass spectrometry (MS/MS) using a Q
Exactive™Plus (Thermo Scientific, Waltham).
Enzyme-linked immunosorbent assay

Commercial ELISA kits (mouse TNF-α, EK0527, Bosterand mouse IL-1β, EK0394, Boster) were used. After diluting the standard, samples were added vertically to eachwell without touching the wall of the well. Then, 50 µl of
enzyme reagent was added, and the plate was sealed witha membrane for incubation at 37 ºC for 30 min. Subsequently, the development solution was added to develop
color, and the reaction was finally terminated. The optical density at 450 nm was analyzed using a spectrophotometer, and the protein concentration was quantified bycomparing the OD of the target proteins with a standard
curve.
Statistical analysis

Data were processed and analyzed using GraphPad Prism
software (version 9.0). All measurements were expressedas the mean ± standard deviation (X ± SEM). Statisticalsignificance was calculated for univariate comparisons
between three or more groups using one-way ANOVA
followed by Tukey’s post-hoc test. P < 0.05 were considered statistically significant.

Results
OXA reduces neurocognitive/mood deficits andhippocampal neuronal apoptosis 1 day after sepsis in adose-dependent manner

To assess whether the CLP-induced sepsis mouse model
exhibited cognitive dysfunction, we performed behavioral tests and evaluated the effects of OXA on any
observed deficiencies. Mice were assessed for cognitive,

Guo et al. Journal of Neuroinflammation(2024) 21:131

motor, and emotional deficits using the OFT. As shownin Fig. 1B, C, there were no significant differences in the
proportion of distance travelled or time spent in the central area between the sham and sham + OXA 500 µg/kg
groups. CLP mice showed a significant decrease in central area dwell time and distance travelled (****p < 0.0001,**** p < 0.0001 vs. sham group), while OXA-treated micein the CLP + OXA 250 µg/kg and CLP + OXA 500 µg/
kg groups had significantly increased central area dwell
time and distance travelled (****p < 0.0001, ***p < 0.001,***p < 0.001, *p < 0.05 vs. CLP group). However, comparedwith the CLP group, the difference was not statisticallysignificant in the CLP + OXA 50 µg/kg group.
Mood disturbance was assessed using the TST. As
shown in Fig. 1D, there were no significant differencesin the proportion of resting time after tail suspension
within 5 min between the sham and sham + OXA 500 µg/
kg groups. CLP mice showed a significant increase in
resting time within 5 min (**p < 0.01 vs. sham group),
OXA-treated mice in the CLP + OXA 250 µg/kg and
CLP + OXA 500 µg/kg groups showed a decreased resting time (*p < 0.05, *p < 0.05 vs. CLP). However, comparedwith the CLP group, the difference was not statisticallysignificant in the CLP + OXA 50 µg/kg group.
The exploratory and memory abilities of mice wereassessed using the Y-maze test. As shown in Fig. 1E, F,
there were no significant differences in the percentage of
exploration time and activity distance within the new arm(C-arm) of the mice between the sham and sham + OXA
500 µg/kg groups. The percentage of exploration timeand activity distance was significantly decreased in CLP
mice (****p < 0.0001, ****p < 0.0001 vs. sham group),
while OXA-treated mice in the CLP + OXA 250 µg/kg
and CLP + OXA 500 µg/kg groups had increased percentage of exploration time and percentage of distance(***p < 0.001, *p < 0.05, **p < 0.01, *p < 0.05 vs. CLP group).
However, compared with the CLP group, the difference
was not statistically significant in the CLP + OXA (50 µg/kg) group.
Pathological changes of neurons in the CA1 area of the
hippocampus were observed by Nissle staining. Overall,neurons in the sham and sham + 500 µg/kg OXA groups
were neatly and compactly arranged, with rounded cells
and obvious rounded nuclei in multilayered formations.
Conversely, neurons in the CLP and low dose OXA(50 µg/kg) groups were vacuolated, disorganized andsparsely arranged with irregular morphology. Neuronaldamage was ameliorated in the OXA medium (250 µg/kg) and high dose (500 µg/kg) groups (Figure S1).
The above results showed that one day after CLP, mice
showed reduced basal motor activity, lack of exploration, increased anxiety-like behavior, and damage to hippocampal neurons. However, intranasal OXA showed
protective effects and the most significant benefit wasPage 5 of 19

observed at a dose of 250 µg/kg. Therefore, future studies
used 250 µg/kg OXA.
Continuous administration of OXA for 7 days was effectiveat reducing mortality and improving cognitive andaffective impairment after sepsis

Based on the results of the post-operative test, the doseof 250 µg/kg was used as the optimal dose for the subsequent test. To understand the effect of 250 µg/kg OXA on
survival within 7 days after sepsis-induced brain injury,
we assessed the mortality rate following daily intranasal
administration OXA 250 µg/kg after CLP. All mice in
the Sham group and the Sham + 250 µg/kg OXA groupsurvived over 7 days. However, the 7-day survival rateof mice in the CLP group was low, with only 28% (8/28
mice) surviving; after treatment with OXA, the survival rate increased to 56% (13/23 mice). We found that
250 µg/kg OXA significantly increased the survival rateof mice with sepsis (Fig. 2A).
To further determine the improvement of 250 µg/kg
OXA on cognitive and emotional deficits in mice after
septic encephalopathy, we performed behavioral tests on
mice 7 days after surgery. In the OFT, as shown in Fig. 2B,C, there were no significant differences in the proportionof distance travelled or time spent in the central region
between the Sham and Sham + OXA 250 µg/kg groups.
Conversely, CLP mice showed a significant decrease in
the time spent in the central region and the distance travelled (****p < 0.0001, ****p < 0.0001 vs. sham group). The
OXA group showed an increase in the distance travelled
and time spent in the central region (*p < 0.05, **p < 0.01,
vs. CLP group).
In the TST, as shown in Fig. 2D, there was no significant difference in resting time after tail suspension
within 5 min between mice in the sham and sham + OXA
250 µg/kg groups. CLP mice showed a significant
increase in resting time within 5 min (***p < 0.001 vs.
sham group), and resting time was decreased in the OXA
group (**p < 0.01 vs. CLP group).
In the Y-maze, as shown in Fig. 2E, F, there were no
significant differences in the percentage of exploration
time and activity distance within the new arm (C-arm)of the mice between the sham and sham + OXA 250 µg/
kg groups. The percentage of exploration time and activity distance were significantly reduced in CLP mice(****p < 0.0001, ****p < 0.0001 vs. sham group). The percentage of exploration time within the new arm and thepercentage of active distance increased in the OXA group(*p < 0.05, **p < 0.01, vs. CLP group).
These results suggested that OXA is effective at reducing mortality and ameliorating cognitive and affective
deficits in postseptic mice, leading us to investigate theunderlying protective mechanisms.

Guo et al. Journal of Neuroinflammation(2024) 21:131

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Fig. 1 OXA ameliorates mood and cognitive deficits and hippocampal neuronal damage 1 day after sepsis in a dose-dependent manner. A Schematic
timeline of the experimental procedures. B Time in the central zone and C distance of activity in the open field. D Resting time within 5 min in the tailsuspension test. E Percentage of time spent exploring and F percentage of distance activity in the C-arm in the Y-maze. G Open field test and Y-mazemovement trajectory maps. The above behavioral tests were performed 1 day after surgery. n = 8 per group. Data are presented as mean ± SEM. *p < 0.05,**p < 0.01, ***p < 0.001, ****P < 0.0001

OXA ameliorates BBB disruption in SAE mice

The BBB ensures normal brain function by maintaining the stability of the brain microenvironment [36]. To
investigate whether OXA had a protective effect on the
BBB, we first examined the degree of cerebral edema.
As shown in Fig. 3A, the brain water content increasedin the CLP group (***p < 0.001 vs. sham group) anddecreased in the OXA group (**p < 0.01 vs. CLP group).
Subsequently, we evaluated the permeability of the BBB.

As shown in Fig. 3B and C, in contrast to the sham group,
the EB content in the brain tissue significantly increasedin the CLP group (***p < 0.001 vs. sham group), and OXA
significantly decreased the EB content (*p < 0.05, vs.
CLP group). IgG in the brain is predominantly secreted
by immune cells that infiltrate the brain; therefore, we
examined the level of IgG in the brain. The data in Fig. 3D
and E show that OXA administration after CLP significantly decreased the area of IgG penetration in the brain

Guo et al. Journal of Neuroinflammation(2024) 21:131

Page 7 of 19

Fig. 2 Treatment with 250 µg/kg OXA increases 7-day survival and ameliorates cognitive and affective deficits in CLP-induced sepsis. A Survival curves.
Values are expressed as the survival rate. Overall, 8 out of 28 mice in the CLP group and 13 out of 23 mice in the OXA group survived to day 7. Surviving
mice were used for subsequent experiments. B Time in the central zone and C distance of activity in the open field. D Resting time within 5 min in the
tail suspension test. E Percentage of time exploration and F percentage of distance activity in the C-arm in the Y-maze. The above behavioral tests were
performed 7 days after surgery. n = 8 per group. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001(**p < 0.01 vs. CLP group). These data indicated that OXA
improved BBB integrity after CLP.
Next, we examined the BBB ultrastructure. Electron
microscopy of brain microvessels in the hippocampusof the mouse brain 7 days after surgery revealed that the
inner layer of the vessel wall in the sham group consistedof endothelial cells, the outer layer consisted of pericytes, and the outermost layer appeared as a footplatefor processes derived from astrocytes surrounding theperiphery (Fig. 3F). In the CLP group, the blood vessels
were significantly constricted and the vessel lumen collapsed. The endfeet of astrocytes attached to the bloodvessels were also significantly swollen. The swollen areas(teals) were bright and hyaline. However, in the OXAtreated group, endothelial cells and basement membrane
structure remained intact, indicating an improvement in
structural damage. We found no obvious differences in
the length or intensity of tight and adherens junctions(TJ/AJ) among the three groups (Fig. 3G). Furthermore,
electron microscopy of astrocytes revealed significant
cellular edema after CLP. Astrocytes with medium-sized
nuclei and homogeneous cytoplasmic structures wereobserved in both the Sham and OXA-treated groups.
However, the cytoplasm of the model group was slightly
stained and appeared hyaline, whereas fragments of different sizes and large and small vesicles due to cellularedema were visible. Significant damage to the organelles
were observed (Figure S2). Overall, these results suggestthat the BBB is disrupted after CLP, but that OXA treatment ameliorates BBB damage.
OXA improves ultrastructural damage in septic mice

We observed the ultrastructural characteristics of thebrains in mice with sepsis to assess the brain protective effects of OXA. The results showed that the endoplasmic reticulum is significantly altered. As shown inGuo et al. Journal of Neuroinflammation(2024) 21:131

Page 8 of 19

Fig. 3 OXA attenuates cerebral edema, protects BBB integrity, and ameliorates ultrastructural damage 7 days after CLP. A Content of cerebral edema. B, C
Extravasation of EB. D, E Extravasation of IgG. OXA treatment reduced extravasation of endogenous IgG into brain tissue. n = 4 per group. F Representativetransmission electron microscopy images showing the BBB ultrastructure. In the CLP group, the edematous astrocyte endfeet (teal) enwrap the vessel.
Scale bar = 2 μm. n = 4 per group. G G is a magnified view of F. Tight junctions of the BBB are highlighted at high magnification. TJ/AJ = tight and adherens
junctions; EC = brain endothelial cells; Acfp = astrocyte foot processes. Scale bar = 1 μm

Fig. 4A and B, the hippocampal endoplasmic reticulumof mice in the CLP group showed significant edema anddilation (***p < 0.001 vs. sham group), which improvedin the OXA-treated group (***p < 0.001 vs. CLP group).
The mitochondria in the CLP group were swollen andthe cristae were weakened or even disappeared, whereas
the mitochondrial structure was improved in the OXA

group (Fig. 4C). The synaptic structure observed using
electron microscopy showed clear gaps between the presynaptic and postsynaptic membranes in the sham group.
However, the CLP group showed significant fusion
between the presynaptic and postsynaptic membranes,
as well as damage and an indistinct diffuse gap in thepostsynaptic membrane. In the OXA-treated group, theGuo et al. Journal of Neuroinflammation(2024) 21:131

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Fig. 4 OXA treatment for 7 days attenuates ultrastructural damage in septic mice. A Representative transmission electron micrograph of three groups ofhippocampal neurons, blue arrow: endoplasmic reticulum. Scale bar = 5 μm. B Quantification of endoplasmic reticulum width in neurons. Values represent the mean of each group. n = 4 per group. C C is a magnified view of A. High magnification of neurons showing details of mitochondria, green arrows:
mitochondria. Scale bar = 2 μm. D Representative electron micrographs of three groups of hippocampal synapses, red arrows: synaptic structures. n = 4
per group. Scale bar = 500 nm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001

Guo et al. Journal of Neuroinflammation(2024) 21:131

presynaptic membrane was partially fused with the postsynaptic membrane, resulting in less damage (Fig. 4D).
These results suggested that treatment with OXA ameliorated the ultrastructural damage induced by CLP in thebrains of mice with sepsis.
OXA reduces septic encephalopathy-induced inflammationand inhibits microglial activation in the cortex andhippocampus of mice

Sepsis is an inflammatory disease that affects the immune
system. The release of inflammatory factors exacerbates
inflammatory response and apoptosis, leading to brain
tissue damage. As shown in Fig. 5A, B, ELISA revealed
a significant increase in TNF-α and IL-1β levels in cortical and hippocampal tissues on both the first and seventh
day after sepsis induction (****p < 0.0001, ****p < 0.0001,****p < 0.0001, ****p < 0.0001 vs. sham group), OXA
treatment significantly reduced the expression levels of
TNF-α and IL-1β (***p < 0.001, **p < 0.01, ****p < 0.0001,*p < 0.05, ***p < 0.001, ****p < 0.0001 vs. CLP group).
Microglial activation mediates neuroinflammationin the brain. Microglial immunoreactivity in the hippocampus and cortex was assessed using Iba-1 staining. As
shown in Fig. 5C and D, immunofluorescence images of
the hippocampus and cortex were taken on the first andseventh days after sepsis, showing a significant increasein the number of Iba-1-positive cells (****p < 0.0001,****p < 0.0001 vs. sham group), while the number ofactivated microglia in the cortex and hippocampusof the OXA-treated group was significantly reduced(****p < 0.0001, ****p < 0.0001 vs. CLP group). Similarly,
magnified images of Iba1-positive cells in septic mice
showed an enlarged cell body and thick processes, consistent with activated microglia (***p < 0.001, **p < 0.01
vs. sham group). In contrast, microglial cells in the OXA
group had thin cell bodies and elongated processes, consistent with the morphology of resting microglial cells(**p < 0.01, *p < 0.05 vs. CLP group).
Proteomic sequencing analysis

To investigate the specific mechanism by which OXA
exerts anti-inflammatory effects in septic brain injury,
we collected hippocampal tissue from the sham, CLP,
and OXA (250 µg/kg) groups on the 7th day after surgery. Samples were subjected to enzymatic digestion
and tandem LC-MS/MS analysis. Bioinformatic tools
were used to perform quantitative proteomic analysis(Fig. 6A), identifying a total of 5680 proteins. To identify proteins that underwent significant dynamic changes
after OXA treatment, we performed expression pattern clustering analysis. Relative protein expression was
first transformed using a Log2 logarithmic transformation. Proteins with an SD > 0.5 were then screened. Afterscreening, the remaining 127 proteins were used forPage 10 of 19

cluster analysis based on their expression patterns. The
number of clusters, k, was set to six, and the degree ofcluster fuzziness, m, was set to two. As shown in Fig. 6B,
the expression pattern classes (clusters) corresponding
to the labelled proteins, with proteins in the same cluster showing similar trends in expression transformation,
were divided into six groups. Statistical analysis revealed
significant upregulation of protein expression levels in
the fourth cluster of proteins after CLP. Furthermore,
it was observed that the protein expression levels were
restored after treatment with OXA. This finding suggeststhat the fourth cluster of proteins might play a criticalrole in the rescue or regulatory effects of OXA treatment.
Therefore, we focused on investigating specific proteins
within this cluster and their relationship with the therapeutic effects of the drug. GO and KEGG analyses ofcluster 4 proteins indicated that their molecular functions were primarily associated with complement andcoagulation cascades, as well as immune response processes. This further suggests that the inhibition of theinflammatory response may be an important mechanism
underlying the treatment of CLP-induced SAE.
Specific KEGG pathways enriched in cluster 4 proteins
included complement and coagulation cascades, malaria,
and MAPK (Fig. 6C). The MAPK pathway is critical forcell proliferation, stress responses, and inflammation. It
activates microglia to release inflammatory factors, leading to inflammation and apoptosis in the central nervous
system [37, 38]. Therefore, we focused on the MAPK
pathway, and the expression of Rras proteins in cluster
4 of the MAPK pathway was quantitatively measured
to create a line graph (Fig. 6D). Rras is a member of thesmall G protein superfamily known as RAS. RAS is a key
protein involved in the MAPK signaling pathway whichacts as a molecular switch interacting with downstream
factors to transduce extracellular signals into the intracellular environment [39]. We examined the expression of
the Rras and the RAS/MAPK axis in the sham, CLP, andOXA groups.
OXA reduces the expression of the Rras protein andinhibits the activation of the RAS/MAPK pathway to reduce
the expression of inflammatory factors

To validate the proteomic results, we examined the protein expression levels of the Rras and the RAS/MAPK
axis in the hippocampal tissues of mice in the sham,
CLP, and OXA groups. Western blot analysis was performed for verification. As shown in Fig. 7A and B, theresults showed that the expression of Rras, RAS, P-JNK
and P-P38 significantly increased after CLP (**p < 0.01,**p < 0.01, ***p < 0.001, ***p < 0.001 vs. sham group), andthe phosphorylation levels of Rras, RAS, P-JNK and
P-P38 significantly decreased after OXA administration(**p < 0.01, **p < 0.01, ***p < 0.001, ***p < 0.001 vs. CLP

Guo et al. Journal of Neuroinflammation(2024) 21:131

Page 11 of 19

Fig. 5 OXA reduces inflammatory cytokine levels and microglial activation in CLP-induced SAE. A, B Expression levels of TNF-α and IL-1β in cortical andhippocampal tissue. n = 6 per group. C Cortex and hippocampus with representative Iba-1 and DAPI immunofluorescence staining. Scale bar = 100 μm.
The upper right corner shows representative magnified Iba-1-positive cells. Scale bar = 20 μm. D, E Whole brain Iba-1 positive cell counts and statisticalanalysis of the cytosolic area of positive cells were performed. n = 4 per group, with 4 sections per animal. Data are presented as mean ± SEM. *p < 0.05,**p < 0.01, ***p < 0.001, ****P < 0.0001

Guo et al. Journal of Neuroinflammation(2024) 21:131

Page 12 of 19

Fig. 6 Proteomic clustering analysis of hippocampal tissue in the mouse brain. A Pattern diagram of the proteomic analysis process. B Clustered protein
expression patterns in the Sham, CLP, and OXA groups. Six clusters were identified by co-expression. Each fold line represents one protein. Left panel:
Co-expression patterns of proteins in the six clusters. Right panel: The top two most significantly enriched entries in each cluster resulting from the enrichment analysis. C KEGG enriched pathway of proteins in cluster 4. D Quantitative expression of Rras proteins in the MAPK pathway in cluster 4. n = 3
per group

Guo et al. Journal of Neuroinflammation(2024) 21:131

Page 13 of 19

Fig. 7 OXA reduces inflammation by decreasing Rras expression and impeding activation of the RAS/MAPK pathway. A, B Representative protein bands
and quantitative analysis of Rras, RAS, P-JNK/JNK, P-P38/P38 and C, D IL-1β, TNF-α in hippocampal tissue 7 days after modelling. n = 3 per group. Data arepresented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

group). As shown in Fig. 7C, D, the expression of downstream inflammatory factors IL-1β and TNF-α was also
increased in the model group (***p < 0.001, ***p < 0.001
vs. sham group), while OXA treatment led to a decreasein the expression of inflammatory factors (*p < 0.05,***p < 0.001 vs. CLP group). Based on the above studies,
we hypothesized that the neuroprotective mechanismof OXA after CLP involves a reduction in inflammation
by decreasing Rras protein expression and inhibiting theactivation of the RAS/MAPK pathway.
Differential effects of OXR1 and OXR2 receptors in theregulation of inflammatory expression through the MAPK
pathway

Exogenous OXA expression in the brain was detected bywestern blotting (Fig. 8A). Protein blot analysis showedthat OXA expression was reduced in the hippocampal

tissue of the mouse brain after CLP (****p < 0.0001 vs.
sham group). However, OXA protein levels significantly
increased after OXA administration (***p < 0.001 vs.
CLP group), and as shown in Fig. 8A, B, the expressionof OXR1 and OXR2 expression was not affected by OXA
treatment.
As shown in Fig. 8C, D, consistent with the findings ofprevious studies, OXA treatment significantly decreased
the expression of P-JNK, P-P38 and the downstream
inflammatory factors IL-1β and TNF-α (**p < 0.01,****p < 0.0001, ****p < 0.0001, ****p < 0.0001 vs. CLP
group). These effects were not observed when a selective OXR1 inhibitor (SB-334,867) was administered.
However, administration of the selective OXR2 inhibitor(JNJ-10,397,049) significantly increased the expression of
P-JNK, P-P38 and the downstream inflammatory factors
IL-1β and TNF-α in mouse hippocampal tissue (*p < 0.05,

Guo et al. Journal of Neuroinflammation(2024) 21:131***p < 0.001, ****p < 0.0001, ****p < 0.0001 vs. CLP + OXA
group).
Furthermore, the anti-inflammatory mechanisms
of OXA and its receptors were evaluated by ELISA. As
shown in Fig. 8E, F, the expression levels of IL-1β, TNF-α
were significantly downregulated in the cortical andhippocampal tissues after OXA treatment (***p < 0.001,

Page 14 of 19****p < 0.0001, **p < 0.01, *p < 0.05 vs. CLP group). The
effects of OXA were significantly reversed by an OXR2
inhibitor (JNJ10397049) (*p < 0.05, ****p < 0.0001,****p < 0.0001, ***p < 0.001 vs. OXA group), but could not
be reversed by an OXR1 inhibitor (SB-334,867). These
results suggest that the anti-inflammatory effect of OXA
on SAE is mediated mainly by OXR2 (Fig. 9).

Fig. 8 Differences in the roles of OXR1 and OXR2. A, B Representative protein bands and quantitative analysis for OXR1, OXR2, and OXA in hippocampal
tissue 24 h after CLP. C, D Representative protein bands and quantitative analysis of P-JNK, P-P38, IL-1β, and TNF-α after administration of SB-334,867 (OXR1
blocker) or JNJ10397049 (OXR2 blocker) 24 h after CLP. n = 3 per group. E, F Levels of IL-1β and TNF-α in brain tissue 24 h after CLP. n = 6 per group. Data
are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001

Guo et al. Journal of Neuroinflammation(2024) 21:131

Fig. 9 Anti-inflammatory signaling pathway mediated by OXA and OXR2
in SAE

Discussion
The pathogenesis of clinical SAE is characterized byaltered mental status, with a wide range of neurologicalpathologies, ranging from delirium to coma, impaired
cognitive performance and consciousness, and episodesof inattention and depression [4, 40]. There is growing
evidence to suggest that the high mortality rate of sepsis
is closely related to acute or long-term cognitive impairment [41, 42]. The hippocampus plays a prominent rolein the cognitive and emotional responses. The CLPmodel is one of the most prominent models for investigating sepsis and related complications, and its ability to
mimic perforated appendicitis in clinical patients makes
it the gold standard of animal models for studying sepsis[29]. Xu [43] and others [44] have both previously conducted behavioral studies in septic mice, stating that themice exhibited cognitive dysfunction and anxiety after
CLP. Consistent with previous studies, we observed that,

Page 15 of 19

on the day after the model was established, the mice
showed obvious erect hair, shortness of breath, purulent
secretions from the eyes, anorexia, and unresponsiveness. Meanwhile, on the seventh day after CLP, the mice
showed obvious autonomous exploration, decreased
spatial memory, and anxiety-like behavior. Expansion of
the endoplasmic reticulum, swelling of mitochondria,
marked disruption of cristae, as well as fusion of presynaptic and postsynaptic membranes, as well as othermanifestations of acute cellular injury in neurons of septic mice were observed by electron microscopy, which
may underlie the pathology of cognitive dysfunction, andevidence from the literature supports such a hypothesis[45–47].
LPS-induced SAE may indirectly inhibit orexigenic
activity through an indirect mechanism [48–50]. Some
investigators have found a 6-fold decrease in appetitive
hormone activity after CLP in mice [51]. These studies
further demonstrated the relevance and importance ofappetitive hormone activity in the pathogenesis of sepsis.
Intranasal (IN) drug delivery has been reported to provide a rapid and direct route to the central nervous system [52, 53], and allows safe and noninvasive targeting of
peptide drugs to the brain [54, 55]. The precise route of
drug delivery from the nose to the brain has been demonstrated, mainly involving the systemic, olfactory, and trigeminal pathways [56, 57], with delivery via the systemicroute exhibiting characteristics similar to intravenous
injection [58, 59]. Studies have reported that intranasal
administration of OXA improves neurofunctional outcomes and reduces cerebral edema following cerebral
hemorrhage [60]. Thus, IN-orexin may be a promising noninvasive treatment strategy for cognitive decline[61]. However, few studies have investigated the effectsof orexin ingestion on cognitive function. In the present
study, we investigated the optimal dosage for effective
OXA treatment of CLP-induced SAE-like brain injury.
We investigated the effects of three different doses of
OXA (50 µg/kg, 250 µg/kg, and 500 µg/kg) on cognitive
function, finding that OXA showed a dose-dependent
improvement in protecting against cognitive impairment
caused by SAE. In addition, we confirmed that treatment
with 250 µg/kg OXA significantly reduced mortality andameliorated cognitive impairment by postoperative day
7 in SAE mice. Overall, we showed that OXA can effectively treat sepsis, and provide an optimal dose of the
drug to ameliorate behavioral deficits.
At the onset of SAE, endothelial cell dysfunction leads
to the impairment of the blood-CSF barrier, resulting
in increased capillary permeability and vasodilation.
This consequently leads to cerebral tissue ischemia andhypoxia [62]. Past studies have shown that BBB permeability increases in the cerebral cortex, hippocampus, andthalamus as early as 24 h after intraperitoneal injection

Guo et al. Journal of Neuroinflammation(2024) 21:131

of LPS [63]. This increase in BBB permeability may leadto the activation of neuroglia and production of cytotoxic mediators that act on the BBB, exacerbating injury.
CLP causes significant cerebral edema, extravasation ofEBs, and elevated IgG levels. IgG in the brain is primarily
produced by the secretion of immune cells that infiltrate
the brain. In this case, the immune system was clearly
activated in sepsis; therefore, the cause of increased IgGlevels was more likely to occur secondary to immune
cell infiltration. Inflammation-induced ultrastructuralchanges in BBB cells remain poorly investigated; however, one of the few other studies investigating ultrastructural changes in the BBB revealed marked edema aroundthe capillaries following LPS-induced injury, but no significant changes in tight junctions [64]. In comparison
with previous research, we did not observe any significant changes in the length and strength of tight junctions
using electron microscopy, but did note significant capillary collapse in the model group. In addition, the swelling of astrocytes in the BBB structure showed extensive
edema; to further confirm this result, we examined astrocytes specifically, finding that the astrocyte endfeet encircle the entire vascular tree within the central nervous
system. These results support a morphological basis for
BBB leakage. Many neurological disorders are characterized by the loss of polarization of specific endfoot proteins, vascular dysregulation, and BBB breakdown. The
role of astrocyte endfeet has been demonstrated in manyof these conditions [65]. Our study fills several knowledge gaps by demonstrating that OXA treatment reverses
sepsis-induced BBB disruption, which may be related to
SAE-induced cerebral vascular endothelial cell dysfunction and reduced vascular endothelial cell inflammation[26, 66]. Amelioration of barrier defects may protect
against SAE-induced neuronal damage [67].
Neuroinflammation is the primary cause of apoptosis
and the subsequent development of brain dysfunction.
Peripheral administration of OXA has been shown to
reduce microglial activation to protect against multiple
sclerosis [68]. In the present study, we observed highexpression of the inflammatory factors TNF-α and IL-1β,
as well as significant activation of microglia in the cortex and hippocampus in brain tissue on days 1 and 7 after
CLP. In addition, prior research has shown that activatedmicroglial cells are capable of producing TNF-α, ultimately leading to neuronal damage [69]. To understandthe inflammatory pathways activated by OXA inhibition,
proteomic analysis was performed to identify the proteins and associated pathways that play critical roles inregulating the anti-inflammatory effects of OXA.
Rras is a member of the Ras superfamily of small
G-proteins, and Ras superfamily proteins regulate
cell proliferation, differentiation and apoptosis [70].
Rras shares approximately 55% amino acid sequence

Page 16 of 19

homology with RAS and there is significant overlap
between Rras and RAS-mediated signaling pathways[28]. Previous studies have shown that mutations in components of the RAS/MAPK pathway cause neurocognitive deficits [71]. In addition, the RAS/MAPK pathway
plays an important role in cancer progression [72, 73]. In
the present study, we confirmed that the protein levels ofRras, RAS, P-JNK, and P-P38 were significantly increasedin the hippocampal tissue after the induction of sepsis ina mouse model of CLP. This suggests that the P-JNK and
P-P38 pathways are activated, confirming the involvement of the MAPK pathway in the development of CLPinduced SAE. However, treatment with OXA significantly
reduced the protein expression of Rras and RAS, as wellas the phosphorylation levels of JNK and P38, resulting
in a decrease in the levels of the downstream inflammatory factors TNF-α and IL-1β. Therefore, it is reasonable
to conclude that OXA reduces the release and production of pro-inflammatory factors by downregulating RAS
expression and inhibiting the P-JNK and P-P38 signaling
pathways.
OXA attenuates cerebral edema induced by cerebral
hemorrhage and reduces neuroinflammation via the
OXR2/AMPK axis [60]. In contrast, in studies of ischemic
stroke, OXA significantly improved neurological deficitscores and reduced inflammatory mediators, primarily
through inhibition of the OXR1/NF-κB pathway [66]. To
gain more insights into the primary pharmacological targets of OXA in the RAS/MAPK pathway in CLP-induced
septic encephalopathy, we performed further experiments using different OXR1 and OXR2 receptor blockers. Our results were validated by western blotting and
ELISA. After testing relevant samples, the OXR1 inhibitor SB-334,867 showed results similar to those of OXA;
however, the OXR2 inhibitor JNJ10397049 significantly
reversed the effects of OXA. This suggests that OXAprimarily binds to OXR2. OXR1 and OXR2 may exert
different effects in different models of OXA binding todifferent receptors. We speculate that these differences in
efficacy may be due to the different models and the activation of different signaling pathways. Many studies haveshown that OXR1 and OXR2 bind to other subunits toform dimers that activate different downstream signaling
molecules [74, 75]. This result confirms our hypothesis.
The orexin system is involved in a variety of physiological functions, including the central regulation of
pancreatic fluid secretion and gastrointestinal motility.
Oku [76] reported that orexin neuropeptides improveintestinal barrier function, and that the brain can control
intestinal permeability. In the present study, we investigated the effect of OXA on the nervous system following
peripheral administration of OXA; however, the effects of
OXA on the intestinal tract and its mechanism were notGuo et al. Journal of Neuroinflammation(2024) 21:131

investigated. This could be an area that could be exploredin depth in subsequent studies.

Conclusions
In summary, we observed that intranasal OXA administration increased the survival rate of septic mice after
seven days, attenuated pathological brain damage andcognitive dysfunction, attenuated BBB damage, inhibitedneuronal apoptosis, and reduced the levels of inflammatory factors. In addition, the proteomic results suggested
that the protective mechanism of OXA may be related to
an induced reduction of Rras and RAS proteins, as wellas the inhibition of MAPK pathway activation. Furthermore, we identified the specific drug targets of OXA thatexert anti-inflammatory effects via the MAPK pathway.
Overall, these results enrich the understanding of thespecific molecular mechanisms underlying the neuroprotective effects of OXA in SAE brain injury, which may
provide promising drug strategies and targets for thetreatment of septic encephalopathy.
Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03111-w.
Supplementary Material 1
Supplementary Material 2
Author contributions
JG conceived and designed the study. JG, ZK, and SY established the animalmodels and performed the behavioral tests. JJD, LZC and GQH analyzed andinterpreted the data. JG wrote and prepared the manuscript. JL, YT, and JQZ
reviewed the manuscript. JQZ coordinated the study. All authors have readand approved the final manuscript.
Funding
This work was supported by the National Natural Science Foundation ofChina (82360376, 81960344, 82360482, and 82260533) and Guizhou Provincial
Science and Technology Projects (ZK[2024]483).
Data availability
All relevant data are included in the manuscript and supplementary files. Any
further inquiries can be directed to the corresponding author.

Declarations
Ethical approval
The animal study was reviewed and approved by the Institutional Animal Careand Use Committee of Guizhou Provincial People’s Hospital (Guizhou, China).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 12 February 2024 / Accepted: 25 April 2024

Page 17 of 19

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