Zhang et al. Journal of Neuroinflammation(2024) 21:186
https://doi.org/10.1186/s12974-024-03185-6

Journal of Neuroinflammation

Open Access

RESEARCH

Lactylation of histone by BRD4 regulates
astrocyte polarization after experimental
subarachnoid hemorrhage
Fan Zhang1,2†, Jian Zhou1,2†, Peng Lu1,2†, Xianhui Zhang2, Lei Yang1,2, Jinpeng Wu1,2, Lihan Zhang2, Lifang Zhang4,
Jinwei Pang1, Huangfan Xie2,3, Bingqing Xie2,3, Yong Jiang1,2,3,4* and Jianhua Peng1,2,5*Abstract
Under subarachnoid hemorrhage (SAH) conditions, astrocytes undergo a marked intensification of glycolytic
activity, resulting in the generation of substantial amounts of lactate to maintain the energy demand for neurons
and other brain cells. Lactate has garnered increasing attention in recent years because of its emerging role incritical biological processes such as inflammation regulation and neuroprotection, particularly through its histonelactylation. Bromodomain-containing protein 4 (BRD4) plays a crucial role in maintaining neural development andpromoting memory formation in the central nervous system. Nonetheless, the function and regulatory mechanismof BRD4 and histone lactylation in astrocytes following SAH remain elusive. Our findings indicate that BRD4, acrucial epigenetic regulator, plays a definitive role in histone lactylation. Both in vitro and in vivo, these results
demonstrated that targeted silencing of BRD4 in astrocytes can significantly reduce H4K8la lactylation, thereby
aggravating the A1 polarization of astrocytes and ultimately affecting the recovery of neural function and prognosis
in mice after SAH. In summary, BRD4 plays a pivotal role in modulating astrocyte polarization following SAH via
histone lactylation. Targeting this mechanism might offer an efficient therapeutic strategy for SAH.
Keywords Subarachnoid hemorrhage, Histone lactylation, Astrocytic polarization, Bromodomain-containing protein
4†

Fan Zhang M.S., Jian Zhou M.S. and Peng Lu M.S. contributed
equally to this work.
*Correspondence:
Yong Jiang
jiangyong@swmu.edu.cn
Jianhua Peng
pengjianhua@swmu.edu.cn
1
Department of Neurosurgery, The Affiliated Hospital, Southwest Medical
University, NO. 25 of Taiping Street, Luzhou, Sichuan 646000, China
2
Laboratory of Neurological Diseases and Brain Function, The AffiliatedHospital, Southwest Medical University, Luzhou, China
3
Institute of Brain Science, Southwest Medical University, Luzhou, China
4
Sichuan Clinical Research Center for Neurosurgery, The AffiliatedHospital, Southwest Medical University, Luzhou, China
5
Academician (Expert) Workstation of Sichuan Province, The AffiliatedHospital, Southwest Medical University, Luzhou, China

Introduction
Subarachnoid hemorrhage (SAH) is a major global publichealth threat, with an annual incidence rate of approximately 6.1 per 100,000 worldwide [1]. Currently, themanagement of SAH primarily emphasizes the treatment of secondary complications [2]. This approach hasprovided a certain degree of assistance in improving thequality of life of patients. With the accelerated aging of
the global population, the public health burden caused by
SAH is becoming increasingly severe [3]. The urgent needto find new brain protection measures is paramount.
Astrocytes play a significant role in neural development, nutritional support, and metabolic regulation in the healthy central nervous system (CNS) [4, 5].

© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified thelicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it.The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation
or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://
creativecommons.org/licenses/by-nc-nd/4.0/.

Zhang et al. Journal of Neuroinflammation(2024) 21:186

However, under acute or chronic adverse conditions,such as acute ischemic stroke (AIS), Alzheimer’s disease(AD) and Huntington’s disease (HD), astrocytes exhibit areactive state and transform into A1 and A2 subtypes [6–9]. In essence, the A1 subtype of astrocytes is harmful, as
they can release proinflammatory cytokines that exacerbate neuroinflammation and ultimately lead to neuronal
death [10]. However, the A2 subtype exerts protective
effects through the upregulation of the expression ofcertain neurotrophic factors. Consistent with our previous findings, blocking A1 polarization of astrocytes after
SAH has neuroprotective effects [11]. Therefore, targeting the phenotypic transformation of astrocytes represents a promising strategy for treating SAH.
The glycolytic pathway in astrocytes abnormally intensifies to sustain the high energy demand of the brain
under pathological conditions [4, 12, 13]. Consequently,
a substantial amount of lactate is generated and transported to neurons via the lactate shuttle, aiming to supply neurons with energy to combat adverse stimuli andmaintain homeostasis [14, 15]. Clinical studies haverevealed that elevated lactate levels in extracellular fluid
and serum following SAH are often predictive of an unfavorable prognosis [16, 17]. Conversely, lactate has been
proposed as a potential therapeutic option for SAH [18].
This controversy may stem from long-standing misunderstandings surrounding the functions of lactate. In
2019, Zhang et al. demonstrated the existence of histonelysine lactylation (Kla). Lactate can promote the transition of macrophages from an inflammatory phenotype
to a homeostatic phenotype through lactylation [19].
Research has discovered that lactate, as a modifying
group, directly binds to the lysine residues of histones,
forming histone lactylation modification, which regulate protein function and promote the transcription of
specific genes. This process has been implicated in themodulation of various diseases, including cancer, myocardial infarction, and Alzheimer’s disease [20–22]. Our
recent research further corroborated the close association between Kla and mitochondrial energy metabolism
following AIS [23].
Bromodomain-containing protein 4 (BRD4), an essential member of the bromodomain and extra-terminal
domain (BET) family, possesses two bromodomains
capable of recognizing lysine residues [24] and is closely
related to various physiological and pathological processes [25]. BRD4 functions as an epigenetic reader for
histone acetylation [24], which is crucial in activatingand elongating gene transcription [26–28]. Lactylation
and acetylation share similar occurrence processes andare regulated by epigenetic factors [29, 30]. However, theassociation between BRD4 and lactylation remains elusive. In the central nervous system, BRD4 is expressedprimarily in neurons and astrocytes, where it plays aPage 2 of 18

pivotal role. Broadly inhibiting BRD4 can alleviate neuroinflammation and oxidative stress after acute ischemic
stroke while maintaining the integrity of the blood-brain
barrier [31]. Conversely, other studies have shown thatloss of BRD4 leads to abnormal development of the neural crest [32] and memory deficits in mice [26]. These
reports suggest the dual nature of BRD4’s function andemphasize the necessity for further exploration. Nonetheless, the involvement of BRD4 in astrocytes following
SAH remains ambiguous.
This study investigated the intricate role of BRD4 andhistone lactylation after SAH. Our findings showed thatlactylation critically regulated astrocyte polarization.
Knocking down BRD4 in astrocytes reduced lactylation,
particularly H4K8la, which in turn exacerbated A1 polarization, increased neuronal death and worsened functional outcomes after SAH. Targeting lactylation and
BRD4 regulation is valuable for the development of neuroprotective drugs.

Materials and methods
Animals

For this study, 161 adult male C57BL/6J mice, aged
between 8 and 12 weeks, weighing 16 to 25 g, andsourced from Chengdu Dashuo Laboratory Animal Co.,
Ltd., were utilized. All animal experiments conductedin this study were approved by the Animal Committeeof the Ethics Committee of Southwest Medical University (Approval Number: 20220223-010). All experimental procedures were performed in accordance with the
National Institutes of Health guidelines for the Care andUse of Laboratory Animals. The mice were housed in acontrolled environment with a 12-hour light/dark cycle,
maintained at a temperature of 22 °C, and had free accessto food and water. The experimental mice were randomly
and blindly assigned to different groups. The animal
experimental design is listed in Supplementary Figure S1.
Cell culture and treatment

The HT22 cell line, derived from mouse hippocampal neurons, was obtained from Procell Life Science &Technology. The C8D1A mouse astrocyte cell line waspurchased from the American Type Culture Collection(ATCC; Rockville, MD, USA). The cells were cultured
in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL
penicillin and 100 µg/mL streptomycin, and incubated ina culture chamber at 37 °C with 5% CO2. Using 10 µM
Oxygenated hemoglobin (OxyHb), dissolved in DMEM,
to stimulate C8D1A cells in vitro to mimic subarachnoid
hemorrhage injury [11]. Solutions of varying concentrations of 2-deoxy-D-glucose (2DG) and lactate (SigmaAldrich, USA) were prepared in DMEM medium forthe purpose of stimulating C8D1A cells. Stable BRD4

Zhang et al. Journal of Neuroinflammation(2024) 21:186

Page 3 of 18

Fig. 1 After 24 h of SAH, astrocytes undergo A1 polarization with elevated levels of BRD4 and lactylation. (A) Representative brain images of sham andSAH mice. (B) mRNA expression of A1 astrocyte markers (C3, Serping1, Gbp2, and Ugt1a1) and A2 astrocyte markers (S100a10, Tm4sf1, Clcf1, and B3gnt5)in mice subjected to sham and SAH conditions (n = 5 or 6). (C-D) Representative fluorescent images and quantification of BRD4 (green) in astrocytes(GFAP, red). Nuclei were counterstained with DAPI (blue, n = 9 fields from 3 mice per group). Scale bar, 50 μm. (E) Lactate levels in the brains of sham andSAH mice (n = 6). (F) Schematic diagram of astrocyte isolation. (G-J) Representative WB images and statistical results of lactylation levels (Indicated by
Pan-Kla) in isolated astrocytes and other cells in sham and SAH mice (n = 10). All values are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns,
no significant changes

knockdown astrocytes (shBRD4) were obtained throughtransfection with BRD4 shRNA (OBiO, China), and Control astrocytes (shCtrl) were treated with control shRNA(OBiO, China). Seventy-two hours after transduction,
the cells were selected with puromycin, followed by theselection of single-clone cells. The target sequence for
BRD4 shRNA was ​T​T​C​T​C​C​G​A​A​C​G​T​G​T​C​A​C​G​T, andthe Control shRNA sequence was ​G​C​G​G​C​A​G​C​T​A​A​G​
T​C​T​A​G​A​T​A​T. The cell experimental design is listed in
Supplementary Figure S1.

Retro-orbital injections of adeno-associated virus (AAV)

The mice were first anesthetized with isoflurane at
3 ml/h for induction and maintained at 1.5 mL/h. Before
the injection, they received ophthalmic local anesthesia via proparacaine hydrochloride eye drops (Alcon,
USA). Using a 29-gauge insulin syringe, 2 × 1011 vg ofastrocyte-targeted AAV (AAV2/PHP. eB-GfaABC1DmCherry-shCtrl/shBRD4-WPRE) was injected into eachmouse. During anesthesia, the body temperature of eachmouse was maintained at a constant level using a warming pad [33, 34]. On the basis of the different types ofviruses injected, the mice were subsequently categorizedZhang et al. Journal of Neuroinflammation(2024) 21:186

Page 4 of 18

Fig. 2 OxyHb promotes A1 subtype astrocytes and increases BRD4 and lactylation. (A) mRNA expression of C3 and S100a10 in astrocytes of the Control
and OxyHb groups (n = 6). (B-D) Representative WB images and quantification of C3 and S100a10 in astrocytes of Control and OxyHb groups (n = 6).
(E) Lactate levels in astrocytes stimulated with OxyHb (n = 6). (F-H) Representative WB images and quantification of BRD4 and Pan-Kla in astrocytes ofControl and OxyHb groups (n = 6). (I-K) Representative fluorescent images and quantification of Pan-Kla (green) and BRD4 (red) expression in astrocytes
after OxyHb treatment. Nuclei were counterstained with DAPI (blue, n = 9). Scale bar, 20 μm. All values are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001, ns, no significant changes

into either the Control group or the BRD4 knockdown(BRD4 cKD) group. Three weeks after AAV transfection, the mice were made available for subsequent
experimentation.
SAH model

The modified single-clip method was used for intravascular puncture to establish an experimental SAH model inmice [35]. Specifically, mice were anesthetized with isoflurane. The target range for body temperature monitoring and maintenance was 36.5–37.5 °C. An incision wasmade along the anterior midline of the neck. The subcutaneous tissue, muscle groups, peripheral nerves, andvascular fascia were carefully dissected and separated.
The right common carotid artery (CCA), external carotid
artery (ECA), and internal carotid artery (ICA) were
exposed. A 6 − 0 monofilament nylon thread was guided
from the ECA to the ICA. Once resistance was felt, thethread was advanced 2 mm further and used to puncture the vessel at the point where the anterior and middle
cerebral arteries diverged. The Sham groups underwent
the same procedure but without vessel puncturing. At

Zhang et al. Journal of Neuroinflammation(2024) 21:186

24 h after SAH, the mice underwent neurological deficit
scoring and other experimental procedures. In Experiments 1 and 2, 16 mice died within 24 h after SAH. In
Experiment 3, one mouse in the SAH-Control group diedwithin 24 h after SAH, whereas two mice in the SAHBRD4 cKD group died, one within 24 h and the otherwithin 48 h. The grouping and mortality data of the miceare listed in Supplementary Table S1.
Behavioral testing

All behavioral tests were conducted by independent
experimenters who were blinded to the group allocationand interventions for testing and analysis.
The Modified Garcia Scale and Beam Balance test wereused to assess acute neurological deficits in the mice following SAH [36–38]. The scoring criteria for the Modified Garcia Scale include spontaneous activity (0–3),
symmetry of limb movement (0–3), movement of forelimbs (0–3), climbing capacity (1–3), touch of the trunk(1–3), and vibrissa touch (1–3). The Beam Balance test
was used to assess the balance and motor ability of themice. The ability of the mice to walk on a wooden beamfor 1 min was scored as: not walk and fall (0); not walk,remains on beam (1); walk, but fall (2); walk less than
20 cm (3); walk beyond 20 cm (4). The Rotarod test wasused to evaluate the motor coordination, balance, andfatigue resistance of the mice. For three days prior tothe official test, the mice were trained on the rotating
rod at 4 rpm/min. On day 4, the rotating rod gradually
increased speed from 4 rpm to 40 rpm over 5 min. The
test concluded if the mouse fell off or grasped the rod
and rotated for 2 rounds without walking on it. The final
motor test data represented the average of three such trials [39]. To evaluate the long-term functional outcomes,
the Open Field test was utilized to assess the spontaneous
locomotor activity of laboratory mice. In brief, before theexperiment, the mice were placed in a quiet, lighted environment and then individually introduced into an openfield arena (50 × 50 × 25 cm) to explore freely for 6–7 min.
Using Smart 3.0 software (Panlab, Spain), their walking distance was analyzed, with a focus on the distancetraveled during the final four minutes [40]. The Morris
Water Maze test was used to assess the spatial learning and memory abilities of the mice 28 days after SAH
injury. The experiment took place in a large, circular pool
filled with opaque water at a temperature of 25 ± 2 °C. The
maze was deliberately divided into four quadrants, oneof which housed a transparent escape platform measuring 10 cm in diameter and situated 1 centimeter beneaththe water surface. During the first five training days, themice were released from one of four starting points andgiven 60 s to swim freely and find the platform. The micethat did not reach the platform within that time were
gently guided there by the experimenter and allowed

Page 5 of 18

to rest on it for 10 s. On the sixth day, the platform wasremoved, and the mice were tested while all other procedures remained the same. The number of times the miceentered the target area were recorded [41].
Isolation of astrocytes from the brain

The mice were anesthetized deeply with pentobarbital
sodium and subsequently sacrificed for brain extraction. Following the instructions of the high-activity brain
enzyme digestion kit (DHABE-5003, RWD, China) andthe single-cell suspension preparation instrument (DSC400, RWD, China), the brain tissue was prepared as asingle-cell suspension. Using Myelin Removal Beads II(130-096-733, Miltenyi Biotec, Germany) and Red Blood
Cell Lysis Buffer (R1010, Solarbio, China), myelin and red
blood cells were removed from the samples. The total cellpellets were subsequently magnetically labeled with FcRBlocking reagent and Anti-ACSA-2 MicroBeads, adhering strictly to the manufacturer’s guidelines. The isolated
astrocytes were utilized for WB or RT-qPCR. The purityof the isolated astrocytes was detected by RT-qPCR (Supplementary Figure S2).
RNA isolation and quantitative real-time PCR (RT-qPCR)

Total RNA was extracted from both cultured cells, isolated astrocytes and tissue utilizing the RNA isolater
Total RNA Extraction Reagent (Vazyme, China) according to the manufacturer’s instructions. A NanoDrop 2000
spectrophotometer (Thermo Fisher Scientific, USA) was
subsequently used to accurately determine the concentration of total RNA in each sample. Afterward, the RNA
samples were converted into cDNA through reverse transcription using the HiScript®III RT SuperMix for qPCR
along with gDNA wiper (Vazyme, China). RT‒qPCR
was subsequently conducted using the Jena qTOWER
real-time PCR system (Jena, Germany) and ChamQTM
Universal SYBR® qPCR Master Mix (Vazyme, China). The
mRNA expression was normalized to β-actin, and the relative expression was quantified using the 2−ΔΔCt method.
The primers used are listed in Supplementary Table S2.
Western blot analysis (WB)

The samples were collected and lysed in radio-immunoprecipitation assay (RIPA) buffer supplemented with aprotease and phosphatase inhibitor mixture (Beyotime,
China). The samples were disrupted using ultrasonication (50% intensity, 5 s, three times), and the reaction
was continued on ice for 30 min. After centrifugation at
13,000 g for 10 min, the supernatant was collected. The
protein concentration was determined using a BCA kit(Beyotime, China), and the proteins were then denatured in SDS-PAGE loading buffer (Beyotime, China) at
100 °C for 10 min. Equal amounts of protein were loaded
onto an SDS-PAGE gel for electrophoresis and thenZhang et al. Journal of Neuroinflammation(2024) 21:186

Page 6 of 18

transferred onto a Polyvinylidene Fluoride (PVDF) membrane. The membrane was blocked with 5% skim milkfor 1 h and incubated overnight at 4 °C with the primary
antibody: anti-BRD4 (1:1000, ab243862, Abcam, UK),anti-GFAP (1:3000, 16825-1-AP, Proteintech, China),
anti-C3 (1:3000, ab200999, Abcam, UK), anti-S100a10(1:1000, MA8362M, Abmart, China), anti-L-lactyl lysine(1:10000, PTM-1401RM, PTM BIO, China, used to measure the level of pan-lysine lactylation (Pan-Kla)), antiL-lactyl-histone H4 (Lys 12) (1;2000, PTM-1401RM,
PTM BIO, China), anti-L-lactyl-histone H4 (Lys 8)(1:2000, PTM1415RM, PTM BIO, China), anti-L-lactylhistone H3 (Lys 9) (1:2000, PTM1419RM, PTM BIO,
China), anti-L-lactyl-histone H4 (Lys 5) (1:2000, PTM1407RM, PTM BIO, China), anti-Histone H3 Rabbit(1:10000, PTM-1001RM, PTMBIO, China), anti-Histone
H3 Mouse (1:10000, MB9211S, Abmart, China), anti-βactin Mouse (1:6000, 66009-1-IG, Proteintech, China),
and anti-β-actin Rabbit (1:6000, 20536-1-AP, Proteintech, China). Then, the membrane was incubated for 1 h
with the appropriate secondary antibody: HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (1:10000,
SA00001-1, Proteintech, China) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (1:10000, SA000012, Proteintech, China). The signal intensity was visualizedusing a chemiluminescence kit (Vazyme, China) and analyzed with ImageJ software (NIH). Raw images of western
blot are listed in Supplementary Figure S9.

UK), and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594)
(1:200, ab150116, Abcam, UK). Subsequently, DAPI was
added to stain the cell nuclei, followed by imaging undera fluorescence microscope with the same experimental
parameters. We specifically focused on the cortex andbrain tissue surrounding the puncture sites.
Cells were seeded onto a polylysine-coated cell climbing slide placed in a 24-well plate and allowed to adhere
to the slide for 24 h before being exposed to OxyHb foranother 24 h. Then, the cells were fixed in 4% paraformaldehyde in PBS at room temperature for 15 min. The cells
were subsequently washed twice with PBS, each time for5 min. Afterward, the cells were treated with 0.1% TritonX 100 at room temperature for 10 min to achieve permeabilization. The subsequent operations were the sameas those described above. The primary antibodies usedwere as follows: anti-BRD4 (1:400, M073541, Abmart,
China), anti-L-Lactyl lysine (1:800, PTM-1401RM, PTM
BIO, China), anti-C3 (1:200, ab97462, Abcam, UK), andanti-S100a10 (1:100, MA8362M, Abmart, China). The
secondary antibodies used were as follows: Goat AntiRabbit IgG H&L (Alexa Fluor® 488) (1:400, ab150077,
Abcam, UK), Goat Anti-Mouse IgG H&L (Alexa Fluor®
488) (1:400, ab150113, Abcam, UK), Goat Anti-Mouse
IgG H&L (Alexa Fluor® 594) (1:400, ab150116, Abcam,
UK). DAPI was added to stain the cell nuclei. Imaging
was performed via fluorescence microscopy under identical experimental conditions.

Immunofluorescence (IF) staining

Astrocytic morphology analysis

Initially, the mice were euthanized via inhalation of an
overdose of isoflurane. Then, the brain tissue was perfused through the heart with PBS and fixed with 4%paraformaldehyde. Following fixation, dehydration was
carried out via a sucrose gradient. Using a CM1850 cryostat from Leica (Wetzlar, Germany), the tissue was precisely sliced into 10 μm thick coronal sections. Before theexperiment, the brain tissue slices were allowed to warmat room temperature for 30 min. After incubation in
rapid antigen retrieval solution for frozen sections (Beyotime, China) for 5 min, the brain tissue sections were permeabilized with 0.3% Triton X-100 for another 10 min.
For 1 h, the brain tissue sections were blocked with 10%
goat serum at room temperature. The sections were subsequently incubated overnight at 4 ℃ with the following primary antibodies: anti-BRD4 (1:200, ab243862,
Abcam, UK), anti-L-lactyl-histone H4 (Lys 8) (1:200,
PTM1415RM, PTM BIO, China), or anti-GFAP (1:500,
3670 S, CST, USA). After being rinsed with PBS on thesecond day, the sections were incubated with the secondary antibody for 1 h. The secondary antibodies usedwere as follows: Goat Anti-Rabbit IgG H&L (Alexa Fluor®
488) (1:200, ab150077, Abcam, UK), Goat Anti-Mouse
IgG H&L (Alexa Fluor® 488) (1:200, ab150113, Abcam,

Under pathological conditions, astrocytes first morphologically change their long, thin processes to more ramified, larger ones with elongated processes [12]. Statisticalanalysis of the number of branches, junctions and endpoints and the length of branches can visually reflect
the reactivity of astrocytes [42]. Using ImageJ software(NIH), in conjunction with the Analyze Skeleton (2D/3D)
plugin, we conducted a quantitative analysis of the morphological alterations in astrocytes within a biologicalsystem. GFAP staining was used to immunolabel the
astrocytes, outlining the contours and skeletons of thecell bodies. Relevant data were obtained through analyses
conducted using Analyze Skeleton (2D/3D) [43].
Terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling (TUNEL)TUNEL staining of brain tissue was performed according to the instructions of the kit (Vazyme, China). After
permeabilization of the brain tissue slices, the samples
were equilibrated with 1x equilibration buffer for 30 min
at room temperature, followed by a one-hour incubation with 50 µL of TdT buffer at 37 °C. The slices were
subsequently counterstained with anti-NeuN (1:500,
ab104224, Abcam, UK) overnight at 4 °C. The next day,

Zhang et al. Journal of Neuroinflammation(2024) 21:186

Page 7 of 18

Fig. 3 The A1 polarization of reactive astrocytes following OxyHb is regulated by lactylation. (A) mRNA expression of GFAP in astrocytes treated with
OxyHb and various concentrations of 2-DG (n = 4). (B-E) Western blot and quantification of Pan-Kla, C3 and S100a10 levels in astrocytes treated with
OxyHb and 4 mM 2-DG (n = 5). (F-H) Representative fluorescent images and quantification of C3 (green) and S100a10 (red) expression in astrocytes after
OxyHb and 4 mM 2-DG treatment. Nuclei were counterstained with DAPI (blue, n = 6, scale bar, 100 μm). (I) mRNA expression of GFAP in astrocytes treatedwith OxyHb and varying concentrations of lactate (n = 4). (J-M) Western blot images and quantification of Pan-Kla, C3 and S100a10 levels after treatment
with OxyHb and 20 mM lactate (n = 6). (N-P) Representative fluorescent images and quantification of C3 (green) and S100a10 (red) in astrocytes after
OxyHb and 20 mM lactate treatment. Nuclei were counterstained with DAPI (blue, n = 6; scale bar, 100 μm). All values are mean ± SD, *P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001, ns, no significant changes

Zhang et al. Journal of Neuroinflammation(2024) 21:186

Page 8 of 18

Fig. 4 Knocking down BRD4 aggravates A1 polarization of astrocytes by reducing H4K8la levels. (A) BRD4 mRNA expression in shCtrl or shBRD4 cells(n = 6). (B, C and E) Western blot showing BRD4 (C) and Pan-Kla (E) expression in shCtrl or shBRD4 cells (n = 6). (D) Intracellular lactate levels in astrocytes
expressing shCtrl or shBRD4 (n = 6). (F) Western blot showing H3K9la, H4K5la, H4K8la, and H4K12la levels in shCtrl or shBRD4 cells (n = 6). (G) BRD4 andH4K8la immunoprecipitation. (H) Intracellular lactate in astrocytes expressing shCtrl or shBRD4 when stimulated with OxyHb (n = 6). (I-L) Western blot
showing H4K8la (J), C3 (K) and S100a10 (L) expression in astrocytes expressing shCtrl or shBRD4 when stimulated with OxyHb (n = 6). (M-O) The levels ofIL-1β, TNF-α, and IL-6 in the ACM (n = 5). (P) Cell viability of neurons cultured with different ACM (n = 6). (Q-R) Representative fluorescent images and quantification of TUNEL+ neurons cultured with different ACM. Nuclei were counterstained with DAPI (blue, n = 6; scale bar = 50 μm). All values are means ± SDs,
*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significant changes

Zhang et al. Journal of Neuroinflammation(2024) 21:186

Page 9 of 18

the corresponding secondary antibody, Goat Anti-Mouse
IgG H&L (Alexa Fluor® 647) (1:200, ab150115, Abcam,
UK), was incubated with the samples for 1 h.
For TUNEL staining of HT22 cells, the cells were first
fixed with 4% paraformaldehyde at room temperature for25 min, and the subsequent operations were the same as
those described above.

4 °C. The levels of inflammatory cytokines in the brain
and ACM were quantified via ELISA kits according to the
manufacturer’s instructions. The results were normalized
to the total protein level of each sample. The results werenormalized separately on the basis of the total protein
concentration in tissues or cell counts.

Lactate assay

The brain tissues were stained with 0.5% cresyl violet
then dehydrated and rinsed with xylene. Nissl bodies anddamaged neurons in the frontal cortex, hippocampus,
and tissues surrounding the puncture site were observed
and photographed.

The lactate concentration in brain tissue and cells wasmeasured using a lactate assay kit (KGT023, KeyGEN
BioTECH, China) according to the manufacturer’s
instructions. The samples were incubated with the working solution for 10 min at 37 °C, the reaction was terminated, and the absorbance was subsequently measured at
530 nm via a spectrophotometer.
Cell viability assay

Using the Cell Counting Kit-8 (HANBIO, China), cell viability was assessed in accordance with the manufacturer’s
guidelines, and the findings were presented as a percentage relative to the viability of control cells.
BRD4 and H4K8la immunoprecipitation (IP)

Total protein was extracted from cultured astrocytes
utilizing a comprehensive lysis buffer mixture. (Celllysis buffer for immunoprecipitation, P0013; Beyotime,
China; protease inhibitor cocktail, HY-K0010; MedChemExpress, USA). Following centrifugation at 13,400 g for
10 min at 4 °C, the protein concentration in the supernatant was measured using a BCA kit. A total of 2,400 µgof protein was incubated with magnetic protein A beads(10002D, Thermo Fisher Scientific, USA) for 30 min
at 4 °C, and then, the supernatant was divided into twoparts and incubated with control IgG (AC005, ABclonal,
China), anti-BRD4 (ab243862, Abcam, UK) and antiL-lactyl-histone H4 (Lys 8) (PTM1415RM, PTM BIO,
China) at 4 °C overnight with constant rotation. The
samples were subsequently combined with fresh protein beads and incubated for 6 h at 4 °C. The beads were
subsequently washed 3 times to remove nonspecificallybound proteins. Finally, the protein bound to the beads
was eluted with 1× loading buffer (FD002, Fude Biological Technology, China) and analyzed by Western blot.
Enzyme-linked immunosorbent assay (ELISA)

The protein levels of IL-1β, TNF-α, and IL-6 were measured via ELISA kits (IL-1β, E-EL-M0037, Elabscience,
China; TNF-α, E-EL-M3063, Elabscience, China; andIL-6, E-EL-M0044, Elabscience, China). Frontal cortex
tissues from identical regions of the right brain hemispheres of Sham and SAH mice were collected, and theastrocyte-conditioned medium (ACM) stimulated with
OxyHB for 24 h was centrifuged at 1000 g for 20 min at

Nissl staining

Statistical analysis

Statistical analysis was performed using GraphPad Prism(Version 9, GraphPad, La Jolla, CA). The Shapiro-Wilk
normality test was used to determine the distributionof the data. The data followed a normal distribution
and were expressed as mean ± standard deviation (SD).
Unpaired 2-tailed Student’s t test was used to compare 2 groups, and one- or two-way analysis of variance(ANOVA) followed by Tukey’s multiple comparisonstest was performed to determine differences among > 2
groups. The data, which were not normally distributed,
were analyzed through either the Mann-Whitney U testor the Kruskal-Wallis test. Statistical significance wasdetermined when p values were < 0.05.

Results
A1 subtype-reactive astrocytes, astrocytic BRD4 expression
and lactylation are increased during the early phase of SAH

To clarify the phenotypic changes in astrocytes after
SAH, we established experimental models of SAH in mice
(Fig. 1A). After 24 h of hemorrhagic injury, as shown by
RT-qPCR, the mRNA levels of markers of A1 astrocytes,
including C3 (Complement 3), Serping1 (Serpin family
G member 1), Gbp2 (Guanylate-binding protein 2) andUgt1a1 (Uridine diphosphate glucuronosyl transferase1a1), were increased. However, S100a10 (S100 calciumbinding proteina10), Tm4sf1 (Transmembrane 4 L six
family member 1), Clcf1 (Cardiotrophin-like cytokine
factor 1) and B3gnt5 (β-1, 3-N-acetylglucosaminyltransferase 5), markers of A2 astrocytes, were expressed at
the same levels as those in the sham group (Fig. 1B). For
the subsequent experiments, we chose C3 and S100a10
as the specific markers for A1 and A2 astrocytes, respectively. Immunofluorescence staining revealed a significant increase in the expression of BRD4 in GFAP+ cells at
24 h post SAH (Fig. 1C-D, Fig. S5A).
Astrocytes are the energy hub that maintains brain
function and are also one of the primary sources of lactate [4]. As shown in Fig. 1E, there was a notable elevation in the lactate levels within the brain tissue after SAH.

Zhang et al. Journal of Neuroinflammation(2024) 21:186

Previous studies have shown that lactylation mediated bylactate plays a crucial role in various physiological processes and diseases. To clarify the changes in lactylationin astrocytes, Magnetic Bead Sorting was conducted toisolate these cells (Fig. 1F). Next, Western blot analysis confirmed that the Pan-lysine lactylation (Pan-Kla)in isolated astrocytes was increased after SAH (Fig. 1GH). However, the lactylation of other cells remained
unchanged (Fig. 1I-J). The above data indicate that astrocytes polarized toward the A1 phenotype after SAH,
accompanied by increased expression of BRD4 and lactylation. On the basis of these findings, we hypothesizedthat BRD4 and lactylation play latent roles in the polarization of astrocytes.
OxyHb induces A1 subtype reactive astrocytes andenhanced the expression of BRD4 and lactylation

To confirm this hypothesis, astrocytes were stimulatedwith 10 µM OxyHb for 24 h [11]. OxyHb enhanced
astrocytic A1 polarization. As shown by PCR and WB(Fig. 2A-D), there was notable upregulation of C3 expression, whereas the expression level of S100a10 remainedlargely unchanged. As expected, OxyHb induced anincrease in lactate levels in astrocytes (Fig. 2E). Moreover, both WB and IF staining indicated that the levelsof lactylation/Pan-Kla and BRD4 were greater than thosein the control groups (Fig. 2F-K). Moreover, as shownin Fig. 2I, BRD4 and Pan-Kla were highly colocalized in
the nuclei of astrocytes. These findings suggest a potential association between BRD4 and lactylation. Throughthese experiments, we found that OxyHb stimulation for
24 h induced A1 subtype reactive astrocytes, increasing
the expression and lactylation of BRD4.
A1 polarization of reactive astrocytes following OxyHb isregulated by lactylation

We next designed experiments to explore the role oflactate and lactylation in the phenotypic polarizationof astrocytes. OxyHb and different concentrations of
2-deoxy-D-glucose (2-DG, a glycolysis inhibitor) wereused to inhibit the production of lactate. These results
revealed that low concentrations of 2-DG had no significant effect, whereas 4 mM 2-DG markedly increased theexpression of GFAP (Fig. 3A). Subsequent experimentswith 4 mM 2-DG revealed that the lactylation of astrocytes was significantly inhibited (Fig. 3B-C), accompanied by a further increase in C3 expression and no change
in S100a10 (Fig. 3B and D-E). When C3 and S100a10
were costained, the results obtained were consistent withthose described above (Fig. 3F-H, Fig. S3A). Conversely,
after the addition of lactate for 24 h, RT-qPCR was used
to detect the mRNA level of GFAP. As shown in Fig. 3I,
the expression of GFAP was downregulated following
lactate treatment, especially in the 20 mM Lac group.

Page 10 of 18

Therefore, we chose 20 mM lactate for further investigation. Remarkably, the expression of lactylation was further upregulated (Fig. 3J-K). Notably, lactate suppressed
the level of C3, which was elevated in response to OxyHb
stimulation (Fig. 3J and L). The impact of lactate treatment on the expression of S100A10 was minimal (Fig. 3J
and M). The immunofluorescence results also revealed
the same changes (Fig. 3N-P, Fig. S3B). These data indicate that increased lactylation can reduce A1 transformation of astrocytes treated with OxyHb.
Knockdown of BRD4 aggravates A1 polarization ofastrocytes by reducing H4K8la levels, subsequently
inducing neuronal death following OxyHb

To investigate the role of BRD4 in regulating astrocytic
polarization following SAH, we generated BRD4 knockdown C8D1A astrocytes. RT-qPCR and WB both confirmed satisfactory gene silencing efficiency (Fig. 4A-C).
The decrease in the intracellular lactate level was minimal, indicating that the glycolytic pathway and lactate
production in the astrocytes were not significantly
affected by BRD4 knockdown (Fig. 4D). Interestingly,
we observed a significant reduction in the level of lactylation after BRD4 interference (Fig. 4B and E). BRD4,an epigenetic regulator, can bind to histone sites such as
H3K9, H4K5, H4K8, and H4K12 [24]. WB revealed thatthe lactylation of multiple histone sites decreased after
BRD4 knockdown, with the most pronounced decrease
observed in H4K8la (Fig. 4F). Through IP experiments,
we validated the direct interaction between BRD4 andH4K8la (Fig. 4G). These results indicate that BRD4 mayfunction in regulating lactylation and H4K8la.
We found that lactylation can regulate the polarizationof astrocytes, whereas BRD4 can modulate lactylation.
We conducted subsequent experiments to explore theeffect of BRD4 knockdown on astrocytic polarization.
As shown in Fig. 4H, the shCtrl and shBRD4 groups presented similar increases in lactate levels upon OxyHb
stimulation, confirming previous results. We conducted
WB to investigate the impact of BRD4 knockdown on
the expression of H4K8la, C3, and S100A10 in astrocytes
stimulated with OxyHb. As shown in Fig. 4I-L, OxyHbshCtrl resulted in significant H4K8la upregulation compared with shCtrl, but no such effect was observed in
shBRD4 or OxyHb-shBRD4. Furthermore, the protein
levels of C3 in the OxyHb-shCtrl group were significantly elevated, and OxyHb-shBRD4 further exacerbatedthese changes. Similarly, the expression of S100A10 was
not affected by the intervention. These results suggestthat BRD4 regulates astrocyte A1 polarization throughH4K8la following SAH.
The release of proinflammatory cytokines and neurotoxic substances by A1 astrocytes can lead to neuronal
death [11]. We measured the content of proinflammatory

Zhang et al. Journal of Neuroinflammation(2024) 21:186

cytokines in the astrocyte-conditioned medium (ACM)of the Control-shCtrl, Control-shBRD4, OxyHb-shCtrl, and OxyHb-shBRD4 groups. The concentrationsof IL-1β, TNF-α, and IL-6 in the ACM of the OxyHbshBRD4 group were the highest (Fig. 4M-O). Then, theHT22 cells were further exposed to the ACM for 24 h.
Cell viability assays and immunocytofluorescence experiments revealed that OxyHb-shBRD4 ACM further
reduced neuronal viability (Fig. 4P) and increased neuronal death (Fig. 4Q-R).
The downregulation of BRD4 enhances the reactivity ofastrocytes in mice after SAH

To corroborate the previous findings, astrocytic BRD4
conditional knockdown (BRD4 cKD) mice were generated by retro-orbital injection with GfaABC1D-targeting
adeno-associated virus 2 (Fig. 5A). Immunofluorescence
was used to verify the signal overlaps between the AAV
and astrocytes (Fig. S4). Astrocytes were isolated fromthe brain, and the BRD4 expression efficiency in these
isolated astrocytes was detected via RT‒qPCR and WB(Fig. 5B-D). The knockdown efficiency was further validated via immunofluorescence in brain slices (Fig. 5E-F,
Figure S5B). Twenty-four hours after SAH, brain lactate
levels were increased in both Control and BRD4 cKD
mice (Fig. 5G). The WB (samples of isolated astrocytes)
and IF results revealed that the expression of H4K8lain the SAH-Control group was greater than that in thesham group, whereas there was no change in the BRD4
cKD group (Fig. 5H-K, Figure S5C). As shown in Fig. 5L
and Figure S5D, astrocytes in the cortex of the punctured
hemisphere were labeled with GFAP (green). SAH-BRD4
cKD exhibited the strongest fluorescence signal intensity (Fig. 5M). The outline and branching structure of theastrocytes were subsequently delineated. Branches, junctions, end-points, and branch lengths were utilized for acomprehensive analysis of the astrocytic reactivity. Similarly, the SAH-BRD4 cKD group demonstrated superior
performance in these key aspects (Fig. 5N). In addition,
the same analysis was performed on astrocytes in thefrontal cortex and hippocampal, and the results obtained
were consistent with those previously results (Fig S6 andS7). These findings suggest that BRD4 plays a pivotal rolein modulating astrocyte reactivity, with a direct correlation with diminished levels of H4K8la.
BRD4 knockdown lead to A1 polarization of astrocytes,
worsening neuroinflammation and neuronal death

Next, we investigated the astrocytic polarization state
by measuring the expression of C3 and S100a10 in the
cerebral hemisphere with puncturing (Fig. 6A). Compared with that in the Sham group, the expression of
C3 increased significantly after SAH, and the increase
was more pronounced when BRD4 was knocked down,

Page 11 of 18

whereas no significant changes in the expression of
S100a10 was detected (Fig. 6B-C). A1 astrocytes produce
abundant proinflammatory cytokines (IL-6, IL-1β, andTNF-α), which exacerbate neuroinflammation following SAH [7]. RT-qPCR revealed that the inflammatory
response was the most severe in SAH-BRD4 cKD mice
(Fig. 6D-F). The changes in these proinflammatory cytokines were also verified at the protein level through ELISA(Fig. 6G-I). In addition, a strong inflammatory response
is unfavorable for neurons. The proportion of TUNEL+
neurons increased in both SAH-Control and SAH-BRD4
cKD mice, with a greater proportion observed in the latter group (Fig. 6J-K, Fig. S5E). To ascertain changes incritical functional regions, we performed Nissl staining
to evaluate the extent of neuronal damage. The results
indicate that varying degrees of neuronal damage wereobserved in the frontal cortex (Fig. S8A-B), hippocampus (Fig. S8C-D), and tissues surrounding the puncturesite (Fig. S8E-F) in the SAH-Control group of mice, andthis alteration was exacerbated in the SAH-BRD4 cKD
group. The survival of neurons is directly correlated with
the neurological function of mice following injury. Compared with the Control group, the cKD group presented
significantly lower Modified Garcia scores and Beam Balance scores after SAH (Fig. 6L-M). These findings indicate that BRD4 knockdown induces A1 polarization ofreactive astrocytes, leading to increased proinflammatory
cytokines and neuronal death after SAH.
Knockdown of BRD4 impaired the recovery of neurological
function after SAH in mice

The recovery of neural function during the chronic
injury stage is crucial for the prognosis of SAH. Twentyeight days after SAH, long-term behavioral tests wereconducted on the mice. During the rotarod test, micein the Control group and BRD4 cKD group were able
to maintain a stable grip on the rotating rod for a relatively long duration without SAH. On the first day after
SAH, the time spent by the mice in the SAH-Control and
SAH-BRD4 cKD groups on the beam was significantly
reduced. Among them, mice in the SAH-BRD4 cKD
group performed the worst. These phenomena were also
observed on the 28th day (Fig. 7A). Additionally, on the
28th day, mice in the SAH-BRD4 cKD group exhibitedthe shortest distance traveled on the rotarod and had thelowest rotational speed (Fig. 7B-C).
Furthermore, we employed the Open Field test to further assess the locomotor and exploratory abilities of themice. After SAH, the mice preferred to stay in the corners of the box rather than freely exploring the entire
space like normal mice (Fig. 7D). In particular, the SAHBRD4 cKD group presented the shortest distance traveled (Fig. 7E). Finally, in the Morris Water Maze test, thelearning and memory abilities of the mice were evaluatedZhang et al. Journal of Neuroinflammation

Fig. 5 (See legend on next page.)(2024) 21:186

Page 12 of 18

Zhang et al. Journal of Neuroinflammation(2024) 21:186

Page 13 of 18(See figure on previous page.)
Fig. 5 Knocking down BRD4 in mouse astrocytes reduces H4K8la, thereby enhancing astrocytic reactivity. (A) Astrocytic BRD4 conditional knockdown(BRD4 cKD) in C57BL/6J male mice was generated via retro-orbital injection of GfaABC1D-targeting adeno-associated virus. (B-F) BRD4 knockdown efficiency in isolated astrocytes was determined via RT-qPCR (B, n = 8), WB (C-D, n = 6) and immunofluorescence (E-F, n = 8 fields from 4 mice; scale bar= 20 μm). (G) Brain lactate concentrations were measured in Control and BRD4 cKD mice following SAH (n = 6). (H-I) Representative WB images and quantification of H4K8la levels in Control and BRD4 cKD mice following SAH (n = 6). (J-K) Representative fluorescent images and quantification of H4K8la levels
in Control and BRD4 cKD mice following SAH (n = 8 fields from 4 mice; scale bar = 20 μm). (L-N) Representative fluorescent images and quantification of
GFAP intensity (M, n = 8 fields from 4 mice; scale bar = 50 μm), sum branches, sum junctions, sum endpoints and total branch length (N, n = 12 astrocytes
from 4 mice; scale bar = 50 μm) of astrocytes in Control and BRD4 cKD mice following SAH. All values are means ± SDs, *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001, ns, no significant changes

by analyzing their trajectories in the circular water maze
(Fig. 7F). Compared with sham mice, SAH-Control
mice showed no significant difference in the number ofcrossings through the target platform, indicating good
recovery. In contrast, SAH-BRD4 cKD mice continued
to exhibit low crossing frequencies, indicating impaired
recovery (Fig. 7G). These data suggest that knocking down BRD4 leads to impaired recovery of learning,memory, locomotion, and balance abilities in mice during
the long-term phase following SAH.

Discussion
In this study, our pivotal discoveries are as follows: (1)SAH triggered A1 polarization in astrocytes and upregulated the expression of BRD4 in astrocytes, which was
accompanied by increased levels of lactate and lactylation. (2) Administration of 2-DG suppressed lactate
production effectively, resulting in a subsequent reduction in astrocyte lactylation and an exacerbation of A1
polarization in OxyHb-stimulated astrocytes. Conversely,
supplementation with lactate increased lactylation andattenuated A1 polarization. (3) Following SAH, BRD4
has emerged as a key modulator of A1 polarization and isstrongly correlated with H4K8la, thereby affecting neuronal survival and the recovery of neural function in mice.
Notably, minimal variations were observed in A2 astrocytes throughout the course of our experiments. A schematic diagram of the mechanism is shown in Fig. 8.
Astrocytes, the most numerous glial cells in the central
nervous system, aid in neuronal survival, growth, andsynapse formation by secreting neurotrophic and growth
factors [5, 44]. Additionally, astrocytes serve as the primary energy source for neurons, ensuring their continued functionality [45]. Under pathological conditions,the astrocyte-neuron lactate shuttle has neuroprotective
effects, protecting neurons from further damage [46, 47].
However, it is imperative to recognize that lactate canalso have a significant effect on astrocytes. Despite extensive research, the intricate relationships among astrocyte
reactivity, astrocyte polarization status, and elevated lactate levels following SAH remain unclear.
We observed an increase in lactylation in astrocytes at 24 h following SAH. Under OxyHb stimulation, treatment with 2-DG inhibited lactate production
and decreased lactylation, which was accompanied by afurther increase in the expression of GFAP and C3. Conversely, the addition of lactate increased lactylation andreduced the expression of GFAP and C3 in astrocytes following SAH, whereas the A2 astrocyte marker S100a10
remained relatively stable throughout these processes.
We did not observe a direct correlation between lactation and A2 astrocytes. The concurrent increase in lactate levels and astrocyte A1 polarization after SAH does
not conflict with our observations. This phenomenon
results from the intricate and complex characteristics ofastrocyte polarization, which is influenced by a range ofcurrently unidentified mechanisms. Notably, the increasein lactate serves to provide energy for nerve cells to copewith adverse environments [14]. In addition, we discovered the regulatory role of lactate in this process.
Previous studies have indicated that increased endogenous lactate can be a factor in poor disease prognosis[16, 17]. However, recent findings have revealed a specific positive role of lactate in brain development, synaptic plasticity and angiogenesis [13, 48, 49]. Lactate not
only serves as a vital energy source but also functions asa metabolic signaling molecule critical to the brain andastrocytes. It plays a central role in energy transportation,
storage, production, and utilization [13]. Moreover, exogenous lactate supplementation has been demonstrated tohave beneficial effects on cerebral metabolism and hemodynamics in patients with traumatic brain injury (TBI)[50]. In ischemic stroke model animals, lactate administration has been shown to attenuate inflammation by
inhibiting the ubiquitination of N-Myc downstream regulated gene 2 (NDRG2) in astrocytes. Additionally, lactate reduces cell death, mitigates weight loss, and lessens
the degree of ischemic damage inflicted [51–54]. We recognized the beneficial effects of lactate on astrocytes andbelieve that the therapeutic potential of lactate after SAH
deserves further investigation.
We also observed a concurrent increase in both BRD4
and lactylation in astrocytes following SAH. Upon subsequent knockdown of BRD4 expression in astrocytes,
we detected a notable decrease in histone lactylation,
particularly in H4K8la levels. BRD4 and H4K8la immunoprecipitation suggested that BRD4 serves a pivotal
regulatory function in H4K8la. There was no significant
decrease in lactate levels in astrocytes after BRD4 knockdown, indicating that BRD4 did not affect astrocyticZhang et al. Journal of Neuroinflammation(2024) 21:186

Page 14 of 18

Fig. 6 Specific knocking of BRD4 in astrocytes boosts A1 astrocyte polarization, triggering inflammatory responses and neuronal death. (A-C) Western
blot showing C3 (B) and S100a10 (C) expression of Control and BRD4 cKD mice after SAH (n = 6). (D-F) mRNA levels of IL-1β, TNF-α, and IL-6 in Control
and BRD4 cKD mice after SAH (n = 5 or 6). (G-I) Protein levels of IL-1β, TNF-α, and IL-6 in Control and BRD4 cKD mice after SAH (n = 6). (J-K) Representative
fluorescent images and quantification of TUNEL+ neurons of Control and BRD4 cKD mice after SAH. Nuclei were counterstained with DAPI (blue, n = 4;
scale bar = 50 μm). (L-M) Short-term neurological function by Modified Garcia score (L) and Beam Balance test (M), n = 10. All values are the mean ± SD,*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significant changes

Zhang et al. Journal of Neuroinflammation(2024) 21:186

Page 15 of 18

Fig. 7 BRD4 knockdown impairs neurological recovery in mice during the chronic phase following SAH. (A-C) Rotarod test was used to assess the falling
latency (A), speed to fall (B), and distance to fall (C) of Control and BRD4 cKD mice after SAH (n = 6). (D-E) Representative trajectory maps and statisticalresults from the Open field test (n = 6). (F-G) Representative trajectory maps and platform crossovers from the Morris Water Maze test (n = 6). All values aremean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significant changes

glycolysis. As mentioned earlier, BRD4, which serves asboth a transcription factor and an epigenetic factor, isinvolved in the regulation of acetylation and methylation[24, 55, 56]. Coincidentally, our results also revealed that
BRD4 performs a comparable regulatory role in lactylation, thereby contributing further to the elucidation of
the diverse functions of BRD4.
The histone H4K8 is highly important in the central
nervous system. Recent studies have firmly establishedthat the increase in MAP-2 expression, achieved throughthe modulation of H4K8 acetylation, can effectively mitigate the harmful effects of cerebral ischemia-reperfusion
injury [57]. H4K8la is considered to be closely related toembryonic development [58]. Our study revealed thatsilencing BRD4 significantly reduced H4K8la levels andexacerbated A1 polarization and inflammatory responses
in astrocytes. The release of neurotoxic substances by A1
astrocytes, which triggers neuronal death, is intricately
associated with the severity and prognosis of SAH [59,60].
The pivotal role of BRD4 in the central nervous system
is beyond question [25]. The absence of BRD4 impairs
critical synaptic proteins, leading to memory deficits inmice [26]. In addition to its neuroprotective role, studies
have reported that inhibiting BRD4 function can alleviate
inflammatory and other adverse reactions [31, 61]. Furthermore, numerous studies have pointed to the beneficial role of BRD4 in maintaining normal functions. BRD4
orchestrates genome folding to promote neural crest differentiation, and BRD4 deletion in the neural crest resultsin cohesinopathy-like phenotypes [32]. Deletion of BRD4
in adult mice leads to acute deterioration of cardiac contractile function [62]. Using an inducible BRD4 knockout
mouse model, researchers have demonstrated that deletion of BRD4 (BRD4Δ/Δ) in the hematopoietic system
impairs hematopoietic stem cell (HSC) self-renewal anddifferentiation, which is associated with H3 clipping [63].
A comprehensive exploration of astrocyte BRD4 function is imperative to further clarify the neuroprotective
molecular mechanisms underlying BRD4 in SAH.
We provided the mice with an optimized living environment and a diet with a scientifically established nutrition formula. We used small surgical incisions, which can
reduce the additional damage. Simultaneously, strictly
monitoring body temperature, respiration, and heart
rate during surgery, positioning the mice correctly andZhang et al. Journal of Neuroinflammation(2024) 21:186

Page 16 of 18

Fig. 8 BRD4-mediated histone lactylation regulates astrocyte polarization after experimental SAH. After SAH, astrocytes undergo polarization toward theneurotoxic A1 phenotype. BRD4 mitigates the A1 polarization of astrocytes via H4K8la. However, when BRD4 is knocked down, H4K8la levels are significantly reduced, subsequently leading to the exacerbation of A1 polarization in astrocytes. This, in turn, enhances the marked release of proinflammatory
factors, including IL-6, IL-1β, and TNF-α, ultimately resulting in neuronal death

maintaining a clear respiratory tract after surgery may
reduce mortality rates when establishing this model.
Notably, there are differences in the fluorescence intensity, number of branches, junction points, endpoints,
and branch length of astrocytes in different brain regions(frontal cortex, hippocampus, and tissues surrounding the puncture site) under Sham and SAH conditions,which may be related to the different functional roles ofastrocytes in these regions. In the future, it is worth conducting further extensive experiments to further investigate this finding.
There are several limitations in our research. First,
our study focused exclusively on both animals and cells,
specifically on astrocytes, and focused on the A1 polarization of astrocytes after BRD4 knockdown. Future
experiments will involve overexpression or similar interventions to further refine and strengthen our findings.
Second, although we have been established that BRD4
regulates astrocyte polarization post-SAH through histone lactylation, the intricacies underlying this process remain mostly unmapped and unexplored. Finally,
our current study was confined to male mice, thereby

potentially introducing constraints in the generalizationof our findings. Therefore, the sex aspect deserves further
exploration and refinement in future studies.
In conclusion, our research shows that BRD4 regulates
lactylation and has a significant effect on the polarizationof astrocytes after SAH. This regulatory mechanism is
intricately linked to H4K8la, suggesting a novel perspective on the dysfunctional state of astrocytes following
SAH. Future investigations will delve deeper into the precise mechanisms by which BRD4 and lactylation regulate
astrocyte polarization, providing us with more nuanced
and precise information that could inform the development of more effective therapeutic strategies.
Abbreviations
AAV Adeno	Associated virus
ACM	Astrocyte-conditioned medium
BRD4	Bromodomain-containing protein 4
B3gnt5	β1, 3-N-acetylglucosaminyltransferase 5
C3	Complement 3
Clcf1	Cardiotrophin-like cytokine factor 1
Elisa	Enzyme-linked immunosorbent assay
GFAP	Glial fibrillary acidic protein
Gbp2	Guanylate-binding protein 2
IL-6	Interleukin 6

Zhang et al. Journal of Neuroinflammation(2024) 21:186

IL-1β	Interleukin-1β
IP	Immunoprecipitation
Pan-Kla	Pan-lysine lactylation
S100a10	S100 calcium-binding protein a10
Serping1	Serpin family G member 1
SAH	Subarachnoid hemorrhage
Tm4sf1	Transmembrane 4 L six family member 1
TNF-α	Tumor necrosis factor α
Ugt1a1	Uridine diphosphate glucuronosyl transferase 1a1

Page 17 of 18

7.
8.
9.
10.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03185-6.
Supplementary Material 1
Author contributions
F.Z., Y.J. and J.H.P. designed the experiments. F.Z., J.Z., P.L., X.H.Z., L.Y., J.P.W.,
L.H.Z., L.F.Z., J.W.P, H.F.X. and B.Q.X. performed the experiments. F.Z., J.Z. and
P.L. analyzed raw data. Y.J. and J.H.P. reviewed the data and made substantial
contributions to improving the studies. F. Z. and J.H.P. wrote the manuscript.
All the authors have contributed to the critical revision of the final manuscript.
Funding
This work was supported by grants from the National Natural Science
Foundation of China (82371310, 82271306), the Sichuan Science andTechnology Support Program (2023YFH0069, 2023NSFSC0028, 2022YFS0615,
2023NSFSC1559, 2022NSFSC1421) and the Scientific Research Project of the
Sichuan Provincial Health Commission (23LCYJ040), and the Youth Foundationof Southwestern Medical University and Southwest Medical University Project(2020ZRQNB072, 2021ZKZD013, 2021LZXNYD-P01, 2023QN014, 2022-JYJ-158).

11.
12.
13.
14.
15.
16.
17.
18.
19.
20.

Data availability
No datasets were generated or analysed during the current study.

21.

Declarations

22.

Ethics approval
All procedures involving research animals were conducted Animal Committeeof the Ethics Committee of Southwest Medical University (Approval Number:20220223-010), and were performed in accordance with the National
Institutes of Health guidelines for the Care and Use of Laboratory Animals.
Competing interests
The authors declare no competing interests.

23.
24.
25.
26.

Received: 21 April 2024 / Accepted: 24 July 2024
27.
28.
References
1. Etminan N, Chang HS, Hackenberg K, et al. Worldwide Incidence of Aneurysmal Subarachnoid Hemorrhage according to Region, Time Period, bloodpressure, and Smoking Prevalence in the Population: a systematic review andMeta-analysis. JAMA Neurol. 2019;76(5):588–97.
2. Claassen J, Park S. Spontaneous subarachnoid haemorrhage. Lancet.
2022;400(10355):846–62.
3. Chen X, Giles J, Yao Y, et al. The path to healthy ageing in China: a Peking
University-Lancet Commission. Lancet. 2022;400(10367):1967–2006.
4. Bonvento G, Bolanos JP. Astrocyte-neuron metabolic cooperation shapes
brain activity. Cell Metab. 2021;33(8):1546–64.
5. Allen NJ, Lyons DA. Glia as architects of central nervous system formation andfunction. Science. 2018;362(6411):181–5.
6. Patani R, Hardingham GE, Liddelow SA. Functional roles of reactive
astrocytes in neuroinflammation and neurodegeneration. Nat Rev Neurol.
2023;19(7):395–409.

29.
30.
31.

32.
33.
34.

Wang C, Li L. The critical role of KLF4 in regulating the activation of A1/
A2 reactive astrocytes following ischemic stroke. J Neuroinflammation.
2023;20(1):44.
Nakano-Kobayashi A, Canela A, Yoshihara T, et al. Astrocyte-targeting therapy
rescues cognitive impairment caused by neuroinflammation via the Nrf2
pathway. Proc Natl Acad Sci U S A. 2023;120(33):e2303809120.
Yun SP, Kam TI, Panicker N, et al. Block of A1 astrocyte conversion by
microglia is neuroprotective in models of Parkinson’s disease. Nat Med.
2018;24(7):931–8.
Guttenplan KA, Weigel MK, Prakash P, et al. Neurotoxic reactive astrocytes
induce cell death via saturated lipids. Nature. 2021;599(7883):102–7.
Zhang L, Guo K, Zhou J, et al. Ponesimod protects against neuronal death by
suppressing the activation of A1 astrocytes in early brain injury after experimental subarachnoid hemorrhage. J Neurochem. 2021;158(4):880–97.
Xiong XY, Tang Y, Yang QW. Metabolic changes favor the activity and heterogeneity of reactive astrocytes. Trends Endocrinol Metab. 2022;33(6):390–400.
Wu A, Lee D, Xiong WC, Lactate Metabolism. Signaling, and function in Brain
Development, synaptic plasticity, angiogenesis, and neurodegenerative
diseases. Int J Mol Sci 2023, 24(17).
Chamaa F, Magistretti PJ, Fiumelli H. Astrocyte-derived lactate in stress disorders. Neurobiol Dis. 2024;192:106417.
Magistretti PJ, Allaman I. Lactate in the brain: from metabolic end-product tosignalling molecule. Nat Rev Neurosci. 2018;19(4):235–49.
Radolf S, Smoll N, Drenckhahn C, et al. Cerebral lactate correlates with early
onset pneumonia after aneurysmal SAH. Transl Stroke Res. 2014;5(2):278–85.
Aisiku IP, Chen PR, Truong H, et al. Admission serum lactate predicts
mortality in aneurysmal subarachnoid hemorrhage. Am J Emerg Med.
2016;34(4):708–12.
Bouzat P, Oddo M. Lactate and the injured brain: friend or foe? Curr Opin Crit
Care. 2014;20(2):133–40.
Zhang D, Tang Z, Huang H, et al. Metabolic regulation of gene expression byhistone lactylation. Nature. 2019;574(7779):575–80.
Xiong J, He J, Zhu J, et al. Lactylation-driven METTL3-mediated RNA m(6)a modification promotes immunosuppression of tumor-infiltrating myeloid
cells. Mol Cell. 2022;82(9):1660–e167710.
Fan M, Yang K, Wang X, et al. Lactate promotes endothelial-to-mesenchymal transition via Snail1 lactylation after myocardial infarction. Sci Adv.
2023;9(5):eadc9465.
Pan RY, He L, Zhang J, et al. Positive feedback regulation of microglial glucose
metabolism by histone H4 lysine 12 lactylation in Alzheimer’s disease. Cell
Metab. 2022;34(4):634–48. e6.
Zhou J, Zhang L, Peng J et al. Astrocytic LRP1 enables mitochondria transfer
to neurons and mitigates brain ischemic stroke by suppressing ARF1 lactylation. Cell Metabol 2024, (36):1–15.
Singh MB, Sartor. G C BET bromodomains as novel epigenetic targets forbrain health and disease. Neuropharmacology. 2020;181:108306.
Liu L, Yang C, Candelario-Jalil E. Role of BET Proteins in inflammation and CNS
diseases. Front Mol Biosci. 2021;8:748449.
Korb E, Herre M, Zucker-Scharff I, et al. BET protein Brd4 activates transcription in neurons and BET inhibitor Jq1 blocks memory in mice. Nat Neurosci.
2015;18(10):1464–73.
Muhar M, Ebert A, Neumann T, et al. SLAM-seq defines direct gene-regulatory
functions of the BRD4-MYC axis. Science. 2018;360(6390):800–5.
Moudgil A, Wilkinson MN, Chen X, et al. Self-reporting transposons enable
simultaneous readout of Gene expression and transcription factor binding insingle cells. Cell. 2020;182(4):992–e100821.
Wang N, Wang W, Wang X, et al. Histone lactylation boosts reparative geneactivation post-myocardial infarction. Circ Res. 2022;131(11):893–908.
Wang X, Fan W, Li N, et al. YY1 lactylation in microglia promotes angiogenesis
through transcription activation-mediated upregulation of FGF2. Genome
Biol. 2023;24(1):87.
Liu L, Yang C, Lavayen BP, et al. Targeted BRD4 protein degradation by dBET1
ameliorates acute ischemic brain injury and improves functional outcomes
associated with reduced neuroinflammation and oxidative stress and preservation of blood-brain barrier integrity. J Neuroinflammation. 2022;19(1):168.
Linares-Saldana R, Kim W, Bolar NA, et al. BRD4 orchestrates genome folding
to promote neural crest differentiation. Nat Genet. 2021;53(10):1480–92.
Yardeni T, Eckhaus M, Morris HD, et al. Retro-orbital injections in mice. Lab
Anim (NY). 2011;40(5):155–60.
Prabhakar S, Lule S, da Hora CC, et al. AAV9 transduction mediated by systemic delivery of vector via retro-orbital injection in newborn, neonatal andjuvenile mice. Exp Anim. 2021;70(4):450–8.

Zhang et al. Journal of Neuroinflammation(2024) 21:186

35. Peng J, Wu Y, Pang J, et al. Single clip: an improvement of the filamentperforation mouse subarachnoid haemorrhage model. Brain Inj.
2019;33(6):701–11.
36. Zhang Y, Yang X, Ge X, et al. Puerarin attenuates neurological deficits via
Bcl-2/Bax/cleaved caspase-3 and Sirt3/SOD2 apoptotic pathways in subarachnoid hemorrhage mice. Biomed Pharmacother. 2019;109:726–33.
37. Nakano F, Kanamaru H, Kawakita F, et al. Epidermal growth factor receptor
mediates neuronal apoptosis after subarachnoid hemorrhage in mice. Stroke.
2023;54(6):1616–26.
38. Tao Q, Qiu X, Li C, et al. S100A8 regulates autophagy-dependent ferroptosis
in microglia after experimental subarachnoid hemorrhage. Exp Neurol.
2022;357:114171.
39. Kim S, Kwon SH, Kam TI, et al. Transneuronal Propagation of pathologic alphasynuclein from the gut to the Brain models Parkinson’s Disease. Neuron.
2019;103(4):627–41. e7.
40. Chung DY, Oka F, Jin G, et al. Subarachnoid hemorrhage leads to early andpersistent functional connectivity and behavioral changes in mice. J Cereb
Blood Flow Metab. 2021;41(5):975–85.
41. Xu P, Tao C, Zhu Y, et al. TAK1 mediates neuronal pyroptosis in early brain
injury after subarachnoid hemorrhage. J Neuroinflammation. 2021;18(1):188.
42. Chandra S, Di Meco A, Dodiya HB, et al. The gut microbiome regulates
astrocyte reaction to Abeta amyloidosis through microglial dependent andindependent mechanisms. Mol Neurodegener. 2023;18(1):45.
43. Young K, Morrison H. Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ. J Vis Exp
2018, (136).
44. Santello M, Toni N, Volterra A. Astrocyte function from information processing
to cognition and cognitive impairment. Nat Neurosci. 2019;22(2):154–66.
45. Belanger M, Allaman I, Magistretti PJ. Brain energy metabolism: focus on
astrocyte-neuron metabolic cooperation. Cell Metab. 2011;14(6):724–38.
46. Sun Y, Wang Y, Chen ST et al. Modulation of the Astrocyte-Neuron Lactate
Shuttle System contributes to Neuroprotective action of Fibroblast Growth
Factor 21. Theranostics. 2020, 10(18):8430–8445.
47. Takahashi S. Neuroprotective function of high glycolytic activity in astrocytes:
common roles in stroke and neurodegenerative diseases. Int J Mol Sci 2021,
22(12).
48. Margineanu MB, Mahmood H, Fiumelli H, et al. L-Lactate regulates the expression of synaptic plasticity and neuroprotection genes in cortical neurons: atranscriptome analysis. Front Mol Neurosci. 2018;11:375.
49. Morland C, Andersson KA, Haugen OP, et al. Exercise induces cerebral
VEGF and angiogenesis via the lactate receptor HCAR1. Nat Commun.
2017;8:15557.
50. Bouzat P, Sala N, Suys T, et al. Cerebral metabolic effects of exogenous
lactate supplementation on the injured human brain. Intensive Care Med.
2014;40(3):412–21.

Page 18 of 18

51. Xu J, Ji T, Li G, et al. Lactate attenuates astrocytic inflammation by inhibiting
ubiquitination and degradation of NDRG2 under oxygen-glucose deprivation
conditions. J Neuroinflammation. 2022;19(1):314.
52. Buscemi L, Price M, Castillo-Gonzalez J et al. Lactate Neuroprotection against
transient ischemic brain Injury in mice appears Independent of HCAR1
activation. Metabolites 2022, 12(5).
53. Horn T, Klein J. Neuroprotective effects of lactate in brain ischemia: dependence on anesthetic drugs. Neurochem Int. 2013;62(3):251–7.
54. Zhao Y, Zhang X, Chen X et al. Neuronal injuries in cerebral infarction andischemic stroke: from mechanisms to treatment (review). Int J Mol Med 2022,
49(2).
55. Mills RJ, Humphrey SJ, Fortuna PRJ, et al. BET inhibition blocks inflammation-induced cardiac dysfunction and SARS-CoV-2 infection. Cell.
2021;184(8):2167–e218222.
56. Nicodeme E, Jeffrey KL, Schaefer U, et al. Suppression of inflammation by asynthetic histone mimic. Nature. 2010;468(7327):1119–23.
57. Ma H, Hu ZC, Long Y, et al. Tanshinone IIA Microemulsion protects against
Cerebral Ischemia Reperfusion Injury via regulating H3K18ac and H4K8ac invivo and in Vitro. Am J Chin Med. 2022;50(7):1845–68.
58. Yang D, Zheng H, Lu W et al. Histone lactylation is involved in mouse oocyte
maturation and embryo development. Int J Mol Sci 2024, 25(9).
59. Liang P, Zhang X, Zhang Y, et al. Neurotoxic A1 astrocytes promote neuronal ferroptosis via CXCL10/CXCR3 axis in epilepsy. Free Radic Biol Med.
2023;195:329–42.
60. Liddelow SA, Guttenplan KA, Clarke LE, et al. Neurotoxic reactive astrocytes
are induced by activated microglia. Nature. 2017;541(7638):481–7.
61. Zhou Y, Gu Y, Liu J. BRD4 suppression alleviates cerebral ischemia-induced
brain injury by blocking glial activation via the inhibition of inflammatory
response and pyroptosis. Biochem Biophys Res Commun. 2019;519(3):481–8.
62. Padmanabhan A, Alexanian M, Linares-Saldana R, et al. BRD4 (bromodomainContaining protein 4) interacts with GATA4 (GATA binding protein 4) togovern mitochondrial homeostasis in adult cardiomyocytes. Circulation.
2020;142(24):2338–55.
63. Yang H, Sui P, Guo Y, et al. Loss of BRD4 induces cell senescence in HSC/HPCs
by deregulating histone H3 clipping. EMBO Rep. 2023;24(10):e57032.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.