Invest Clin 66(2): 191 - 204, 2025 https://doi.org/10.54817/IC.v66n2a06

Corresponding author: Wei Huang, Neurology Department, The Third people’s Hospital of Yunnan Province, 292#

Beijing Road, Guandu District, Kunming City, Yunnan Province 650000, China. Email: huangweidr@yeah.net

The p300-NF-κB pathway induces the

activation of the NLRP3 inflammasome

and the pyroptosis of neurons in an in vitro

model of Alzheimer’s disease.

Fengqin Sun and Wei Huang

Neurology Department, The Third People’s Hospital of Yunnan Province, China.

Keywords: Alzheimer’s disease; p300; pyroptosis; NLRP3 inflammasome.

Abstract. Inflammation-induced neuronal death is the primary cause of

Alzheimer’s disease (AD). p300 plays an important role in brain disorders. How-

ever, the role of p300 in AD remains unclear. This study aimed to investigate the

potential of p300 in an in vitro model of AD. Protein expression was detected

using western blotting. The mRNA levels were determined by reverse transcrip-

tion-quantitative polymerase chain reaction. Cytokine release was detected us-

ing an enzyme-linked immunosorbent assay. Cellular function was determined

using the cell counting kit-8, lactate dehydrogenase, and flow cytometry assays.

Chromatin immunoprecipitation and luciferase assays verified the interaction

between nuclear factor kappa B (NF-κB) and the NLR family pyrin domain con-

taining 3 (NLRP3). E1A binding protein p300 (p300) was overexpressed in the

Aβ1-42 induced AD model in vitro. However, treatment with the p300 inhibitor

(GNE-049) alleviated inflammation and Aβ1-42-induced pyroptosis in the neu-

rons. p300 activates NF-κB, which antagonizes the effects of GNE-049 and pro-

motes neuronal pyroptosis. Moreover, NF-κB epigenetically activates the NLRP3

inflammasome. The p300/NF-κB pathway promotes neuronal pyroptosis in an in

vitro AD model by activating the NLRP3 inflammasome. Therefore, the p300/

NF-κB/NLRP3 signalling pathway may be a potential therapeutic target for AD.

192 Sun and Huang

Investigación Clínica 66(2): 2025

En el modelo in vitro de la enfermedad de Alzheimer, la vía

p300 NF - Kappa B induce la activación del inflamasoma NLRP3

y la piroptosis neuronal en un modelo in vitro de la enfermedad

de Alzheimer.

Invest Clin 2025; 66 (2): 191 – 204

Palabras clave: enfermedad de Alzheimer; P300; muerte por quemadura celular; cuerpo

inflamatorio nlrp3.

Resumen. La muerte neuronal inducida por la inflamación es la principal

causa de la enfermedad de Alzheimer (AD). El p300 juega un papel impor-

tante en las enfermedades cerebrales. Sin embargo, se desconoce el papel del

p300 en la AD. El objetivo de este estudio es explorar el potencial del p300 en

modelos in vitro de AD. Se utilizó Western blot para detectar la expresión de

proteínas. Los niveles de ARNm se determinaron mediante la reacción cuanti-

tativa en cadena de la polimerasa de transcripción inversa. Se utilizó la prueba

de inmunoabsorción enzimática para detectar la liberación de citocinas. La

función celular se determinó mediante el contador celular Kit - 8, la lactato

deshidrogenasa y la medición con citometría de flujo. La interacción entre el

factor nuclear Kappa b (nf - Kappa b) y el dominio Pirin 3 (nlrp3), que contiene

la familia NLR, fue verificada por inmunoprecipitación de cromatina y detec-

ción de luciferasa. La proteína de unión a E1A p300 (p300) está sobreexpresada

en un modelo de AD inducido por Aβ1-42. Sin embargo, el tratamiento con un

inhibidor del p300 (GNE - 049) redujo la inflamación y redujo la muerte por

piroptosis neuronal inducida por Aβ1-42. El p300 activa NF - Kappa b, que inhibe

el efecto del GNE - 049 y promueve la muerte por piroptosis neuronal. Además,

NF-κB epigeneticamentelly activa el NLRP3 inflamasoma Epigenética NF-Kappa

B activa los cuerpos inflamatorios nlrp3. La vía p300 / NF - Kappa B promueve

la muerte focal neuronal en modelos in vitro de AD activando el inflamasoma

NLRP3. Por lo tanto, la transmisión de la señal p300/NF-Kappa B/NLRP3 puede

ser un objetivo terapéutico potencial para la AD.

Received: 08-03-2025 Accepted: 10-05-2025

INTRODUCTION

Alzheimer’s disease (AD) is a common

neurodegenerative disorder 1. The symptoms

include apraxia, agnosia, aphasia, and emo-

tional disturbance 2. AD is the leading cause

of dementia and accounts for > 60% of all

cases 3. Currently, nearly 50,000,000 people

suffer from AD worldwide 4. In China, among

the 15.07 million people (≥60 years old)

with dementia, 9.83 million (65.2%) were di-

agnosed with AD 5. Moreover, the incidence

of AD is increasing with an aging popula-

tion6. However, the pathogenesis of AD is

complex, and there are no effective prognos-

tic biomarkers 7. Therefore, identifying the

potential molecular mechanisms underlying

AD may provide novel therapeutic strategies.

Pyroptosis is a form of programmed

cell death characterized by inflammasomes8.

Effect of the p300/NF-kB pathway in an in vitro model of Alzheimer’s disease 193

Vol. 66(2): 191 - 204, 2025

The NLR family pyrin domain containing 3

(NLRP3) is the main inflammasome expressed

in brain tissues 9. Stimuli-induced activation

of NLRP3 inflammasomes cleaves caspase1 10.

The cleaved caspase-1 then cleaves gasdermin

D (GSDMD) and induces the accumulation of

the N-terminus of GSDMD (GSDMD-N), which

drives GSDMD to move to the cell membranes

11. The enrichment of GSDMD-N in the cell

membranes contributes to pore formation

and subsequent relapse, releasing interleukin

(IL)-1β and IL-18 12,13. The activation of the

NLRP3 inflammasome is frequently observed

in patients with AD. The NLRP3 inflammasome

mediates neuroinflammation, cell senescence,

and loss of neurons, which are key causes of AD

14-16. However, inhibition of the NLRP3 inflam-

masome restores neuronal function and allevi-

ates AD development 17.

Nuclear phosphoprotein E1A binding

protein p300 (p300) is an acetyl transfer-

ase 18. p300 regulates numerous biological

processes such as proliferation, autophagy,

apoptosis, and pyroptosis 19,20. Increasing

evidence has suggested that p300 is abnor-

mally expressed in patients with brain disor-

ders. For instance, p300 is downregulated in

ischemia/reperfusion injury, whereas over-

expressed p300 enhances the anti-apoptotic

effects of myocardin-related transcription

factor A 21. However, p300 deficiency in-

hibits neuroepithelial cell proliferation in

diabetes-induced tube defects 22. Moreover,

high levels of p300 in human brains with AD

contribute to neuronal loss 23. However, the

role of p300 in AD has not yet been com-

pletely elucidated. In the present study, we

investigated the potential role of p300 in an

in vitro model of AD. We hypothesized that

p300-mediated neuronal pyroptosis exacer-

bates the progression of AD.

MATERIALS AND METHODS

Cell culture

The mouse neuronal cell lines HT22 and

HEK293T were obtained from ATCC (Manas-

sas, VA, USA). The cells were cultured in

Dulbecco’s Modified Eagle’s Medium supple-

mented with 10% fetal bovine serum (FBS).

The cells were incubated at 37°C in a 5% CO2.

HT22 cells were exposed to Aβ1-42 (20

μM) and a p300 inhibitor (GNE-049, 500

nM) for 24 h. Cells in the control groups

were cultured with FBS.

HT22 cells were transfected with small

hairpin RNA shRNA) of nuclear factor kap-

pa B (NF-κB) and overexpression plasmids

or the control/vector using Lipofectamine

3000 (Invitrogen) according to the manu-

facturer’s instructions.

Enzyme-linked immunosorbent assay

(ELISA) assay

Cytokine levels were measured using

ELISA kits (Abcam, Cambridge, USA), includ-

ing IL-6, IL-1β, IL-18, tumor necrosis factor

(TNF)-α, interferon (IFN)-γ, and IL-10.

Lactate dehydrogenase (LDH) assay

The release of LDH was determined

using the corresponding LDH kit (Abcam,

Cambridge, USA).

Reverse transcription-quantitative

polymerase chain reaction (RT-qPCR)

Total RNA was extracted from the HT22

cells. A HiScript II 1st Strand cDNA Synthe-

sis Kit (Vazyme, China) was used to synthe-

size the cDNA. PCR was performed using the

HiScript II One-Step RT-PCR Kit (Vazyme,

China) on an ABI 7900 system. Glyceralde-

hyde-3-phosphate dehydrogenase served as

the loading control. The mRNA levels were

calculated using the 2−ΔΔCT method.

Western blot

HT22 cells were harvested, and total

protein was extracted. After centrifugation at

12000 × g, a BCA assay was performed to de-

termine the protein concentration. Forty mi-

crograms of protein were isolated using 10%

Sodium dodecyl sulphate-polyacrylamide gel

electrophoresis. Proteins were transferred

to polyvinylidene fluoride membranes. After

sealing with 5% skim milk, the membranes

194 Sun and Huang

Investigación Clínica 66(2): 2025

were incubated with primary antibodies, in-

cluding anti-p300 (ab275378; 1: 1000, Ab-

cam, UK), anti-p-p65 (ab32536; 1: 2000,

Abcam, UK), and anti-GAPDH (ab9485; 1:

2500, Abcam, UK) and then with goat anti-

rabbit IgG H&L (ab205718; 1: 10000, Ab-

cam, UK). Subsequently, the bands were im-

aged using an enhanced chemiluminescence

kit (6104-58-1; Sigma-Aldrich, Germany).

Luciferase assay

JASPAR (https://jaspar.elixir.no/) was

used to predict the binding sites between

NF-κB and the promoter of NLRP3. Bind-

ing was amplified and inserted into the

pMIR-GLOTM luciferase vector (Promega).

HEK293T cells were transfected with wild-

type (WT)/mutant type (MUT) of NLRP3

and NF-κB shRNA/overexpression plasmids.

After 48 h, luciferase activity was detected

using a kit (Promega).

Chromatin immunoprecipitation (ChIP)

assay

A ChIP assay was conducted on HT22 cells

using a ChIP kit (Sigma-Aldrich). Briefly, cells

were crosslinked with 1% formalin, afterwards,

cells were lysed and sonicated. The sonicated

chromatin was incubated with antibodies, in-

cluding anti-NF-κB (ab32536; 1: 30, Abcam,

UK) and anti-IgG (ab172730; 1: 50, Abcam,

UK) using Protein G magnetic beads. Finally,

the DNA fragments were analyzed by RT-qPCR.

Cell counting kit-8 (CCK-8) assay

The cells were seeded in a 6-well plate

(4000 cells/well) and cultured for 0, 24, 48,

and 72 h. The cells were then supplemented

with CCK-8 reagent. Finally, cell viability was

determined using a microplate reader at an

absorbance of 450 nm.

Flow cytometry

Neuronal pyroptosis was detected using

flow cytometry with propidium iodide (PI)

and caspase-1 staining. Briefly, neurons were

digested with ethylenediamine tetraacetic

acid-free trypsin. Then, the cells were har-

vested by centrifugation at 1000 rpm for 5

min. Afterwards, cells were resuspended and

washed with PBS twice. The cells were in-

cubated in the dark with FAM FLICA™ Cas-

pase-1 Kit (ICT098; Bio-Rad, USA) and PI (4

μL). The results were analyzed using a flow

cytometer (Biosciences, USA).

Statistical analysis

Each independent experiment was per-

formed in triplicate. Graphpad v.8. software was

used to analyze the data. Data are presented

as the mean±SD. Student’s t-test and ANOVA

were used to analyze differences. p<0.05 was

considered statistically significant.

RESULTS

p300 is upregulated in an in vitro model

of AD

p300 is frequently upregulated in pa-

tients. Therefore, we determined the p300

expression in an AD model in vitro. We found

that p300 mRNA expression in HT-22 cells

exposed to Aβ1-42 was markedly increased

compared with that in the control group

(Fig. 1A). This finding was consistent with

the Western blot results. Aβ1-42 treatment

markedly increased the protein expression

of p300 (Fig. 1B).

p300 deficiency inhibits

neuroinflammation

p300 is a key regulator of the inflam-

matory response and mediates cerebral

injury by activating inflammation-related

signalling. Therefore, we hypothesized that

p300 promotes AD pathogenesis by inducing

neuroinflammation. As shown in Fig. 2A-E,

the release of proinflammatory cytokines,

such as IL-6, IL-1β, IL-18, tumor necrosis

factor (TNF)-α, and interferon (IFN)-γ, was

significantly increased after Aβ1-42 exposure,

whereas IL-10 was markedly decreased (Fig.

2F). However, GNE-049 treatment signifi-

cantly alleviated the effects of Aβ1-42, inhib-

ited the release of IL-6, IL-1β, IL-18, TNF-α,

and IFN-γ, and increased the release of IL-

Effect of the p300/NF-kB pathway in an in vitro model of Alzheimer’s disease 195

Vol. 66(2): 191 - 204, 2025

Fig. 1. The expression of p300 in in vitro model of AD.

(A) RT-qPCR was conducted to detect p300 mRNA expression in HT-22 cells exposed to Aβ1-42. (B)

Western blot was conducted to detect p300 protein expression in HT-22 cells exposed to Aβ1-42. The

difference in comparison was analyzed using the Student t-test. AD: Alzheimer’s disease; p300:

E1A binding protein p300; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; RT-qPCR: reverse

transcription-quantitative polymerase chain reaction. **p<0.01.

Fig. 2. p300 deficiency inhibits neuroinflammation.

(A-F) ELISA was used to detect the release of cytokines in HT-22 cells. (G-L) RT-qPCR was conducted to detect

cytokine mRNA expression in HT-22 cells. Comparison difference was analyzed using one-way ANOVA. p300:

E1A binding protein p300; IL-6: interleukin 6; IL-1β: interleukin 1β; IL-18: interleukin 18, TNF-α: tumor

necrosis factor α; IFN-γ: interferon γ; IL-10: interleukin 10. ELISA: enzyme-linked immunosorbent assay;

RT-qPCR: reverse transcription-quantitative polymerase chain reaction. **p<0.01, ***p<0.001.

196 Sun and Huang

Investigación Clínica 66(2): 2025

10. This finding is consistent with the RT-qP-

CR results. GNE-049 treatment suppressed

the mRNA expression of IL-6, IL-1β, IL-18,

TNF-α, and IFN-γ (Fig. 2 G-K), while increas-

ing IL-10 mRNA expression (Fig. 2 L). These

findings suggest that the inhibition of p300

expression suppresses neuroinflammation.

p300 deficiency inhibits the pyroptosis

of neurons

Inflammation-induced pyroptosis,

which is characterized by the activation of

the inflammasome and an increase in cy-

totoxicity and death, is a key cause of AD.

p300 protein expression was markedly re-

duced by GNE-049 treatment (Fig. 3A). Aβ1-

42 exposure markedly increased LDH release

(Fig. 3B), which was antagonized by GNE-

049 treatment. Moreover, GNE-049 treat-

ment promoted neuronal viability (Fig. 3C).

GNE-049 treatment markedly alleviated the

pyroptosis in the neurons induced by Aβ1-42

exposure (Fig. 3D). Additionally, GNE-049

treatment suppressed the mRNA expression

of NLRP3, PYD and CARD domain contain-

ing (ASC), and caspase-1 (Fig. 3E). These

findings suggested that p300 inhibition al-

leviates neuronal pyroptosis in AD.

Fig. 3. p300 deficiency inhibits the pyroptosis of neurons.

(A) p300 protein expression was detected using Western blot. (B) LDH assay was conducted to detect

cytotoxicity of HT-22 cells. (C) CCK-8 was performed to determine the cell viability of HT-22 cells.

(D) Flow cytometry was used to detect the pyroptosis of HT-22 cells. (E) RT-qPCR was conducted to

detect mRNA expression in HT-22 cells. Comparison difference was analyzed using one-way ANOVA.

p300: E1A binding protein p300; NLRP3: NLR family pyrin domain containing 3; ASC: PYD and CARD

domain containing; LDH: lactate dehydrogenase; RT-qPCR: reverse transcription-quantitative polyme-

rase chain reaction. **p<0.01, ***p<0.001.

Effect of the p300/NF-kB pathway in an in vitro model of Alzheimer’s disease 197

Vol. 66(2): 191 - 204, 2025

p300 activates NF-κB signaling

p300 participates in the inflammatory

response by activating inflammatory signal-

ling. Therefore, we hypothesized that p300

mediates neuroinflammation by activating

NF-κB signaling. Aβ1-42 exposure significantly

increased the protein expression of p-p65

(Fig. 4), which GNE-049 alleviated.

p300 induces neuroinflammation via

activating NF-κB signaling

To verify the role of NF-κB in AD,

neurons were transfected with an NF-κB-

overexpression plasmid. We found that over-

expression of NF-κB alleviated the effects of

GNE-049 and promoted the release of IL-6,

IL-1β, IL-18, TNF-α, and IFN-γ, as well as

decreased IL-10 (Fig.5A-F). Moreover, over-

expression of NF-κB markedly increased

the mRNA expression of IL-6, IL-1β, IL-18,

TNF-α, and IFN-γ (Fig. 5G-K), but decreased

IL-10 mRNA expression (Fig. 5L).

p300 induces pyroptosis via activating NF-

κB signaling

A rescue assay was conducted to con-

firm further the role of p300/NF-κB signal-

ing in AD. We found that overexpression

of NF-κB markedly alleviated the effects of

GNE-049 and promoted neuronal cytotox-

icity (Fig. 6A). However, NF-κB overexpres-

sion suppressed neuronal viability (Fig. 6B).

Overexpression of NF-κB alleviated the ef-

fects of GNE-049 and promoted pyroptosis

in neurons (Fig. 6C). Moreover, overexpres-

sion of NF-κB markedly increased the mRNA

expression of NLRP3, ASC, and caspase-1

(Fig. 6D). These findings suggest that p300

regulates neuronal pyroptosis by activating

NF-κB signaling.

p300-dependent activation of NF-κB

epigenetically activates NLRP3.

NF-κB, a key transcription factor in

inflammatory signalling, participates in

biological processes via its downstream reg-

ulation. We found that NLRP3 mRNA expres-

sion was markedly increased by p300 overex-

pression and returned to normal levels after

transfection with NF-κB shRNA (Fig. 7A).

NF-κB regulates its downstream activity by

binding to the promoters of its target genes.

Therefore, we hypothesized that NF-κB binds

to the NLRP3 promoter (Fig. 7 B). Fig. 7C

shows the binding motif for NF-κB. Four

binding sites were identified in the promoter

of NLRP3 (Fig. 7c). Overexpression of p300

and NF-κB markedly enhanced the transcrip-

tion of NLRP3 (Fig. 7D). To identify the site

that binds NF-κB, the 3’-UTR of the binding

Fig. 4. p300 activates NF-κB signaling.

Western blot was performed to detect the p-p65 protein expression in HT-22 cells. Comparison diffe-

rence was analyzed using one-way ANOVA. p300: E1A binding protein p300; GAPDH: glyceraldehyde-

3-phosphate dehydrogenase. **p<0.01, ***p<0.001.

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Investigación Clínica 66(2): 2025

sites was mutated and inserted into lucifer-

ase reporters. Co-transfection with p300 and

NF-κB significantly increased luciferase ac-

tivity (Fig. 7E). Moreover, luciferase activity

was markedly increased in MUT1/3/4 cells

after Aβ1-42 exposure, which was antagonized

by GNE-049 treatment (Fig. 7F), whereas

there was no significant alteration in MUT2.

Additionally, p300 deficiency markedly

suppressed the co-occupancies of site2 in

HEK293T cells (Fig. 7G-H). These findings

suggest that the p300-mediated activation

of NF-κB epigenetically upregulates NLRP3

expression.

Fig. 5. p300 induces neuroinflammation via activating NF-κB signalling.

(A-F) ELISA was used to detect the release of cytokines in HT-22 cells. (G-L) RT-qPCR was performed

to detect the p300 mRNA expression in HT-22 cells. Comparison difference was analyzed using one-

way ANOVA. p300: E1A binding protein p300; IL-6: interleukin 6; IL-1β: interleukin 1β; IL-18: inter-

leukin 18, TNF-α: tumor necrosis factor α; IFN-γ: interferon γ; IL-10: interleukin 10. ELISA: enzyme-

linked immunosorbent assay; RT-qPCR: reverse transcription-quantitative polymerase chain reaction.

*p<0.05, **p<0.01, ***p<0.001.

Effect of the p300/NF-kB pathway in an in vitro model of Alzheimer’s disease 199

Vol. 66(2): 191 - 204, 2025

DISCUSSION

In this study, p300 was upregulated in

an in vitro model of AD. Interestingly, p300

deficiency inhibits neuroinflammation and

suppresses pyroptosis in the neurons. More-

over, p300 activates NF-κB, and its overex-

pression promotes pyroptosis in neurons.

Additionally, the p300-mediated activation

of NF-κB epigenetically upregulates NLRP3,

which induces pyroptosis in neurons. There-

fore, the p300/NF-κB/NLRP3 pathway may

be a potential target in AD.

p300 is aberrantly expressed in several

brain disorders. Chatterjee et al.24 revealed

that CBP/p300 activation enhances neuro-

Fig. 6. p300 induces pyroptosis via activating NF-κB signaling.

(A) The LDH assay detected cytotoxicity in HT-22 cells. (B) The CCK-8 assay was performed to deter-

mine the viability of HT-22 cells. (C) Flow cytometry was used to detect pyroptosis in the HT-22 cells.

(D) RT-qPCR was performed to detect the mRNA expression in HT-22 cells. Comparison difference was

analyzed using one-way ANOVA. p300: E1A binding protein p300; NLRP3: NLR family pyrin domain

containing 3; ASC: PYD and CARD domain containing; LDH: lactate dehydrogenase; RT-qPCR: reverse

transcription-quantitative polymerase chain reaction. **p<0.01, ***p<0.001.

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Investigación Clínica 66(2): 2025

genesis and prolongs memory duration, and

maturation, and differentiation of adult neu-

ronal progenitors. However, hyperactivation

of p300 contributes to tauopathy pathogen-

esis 25. Therefore, p300 may play protective

and passive roles in brain disorders. This may

be because the roles of p300 vary with dis-

ease subtype and signalling. Therefore, it is

crucial to identify the role of p300 in AD,

which is very important. CBP/p300 activa-

tion-mediated acetylation of tau exacerbates

traumatic brain injury, which is the most

significant non-genetic, non-aging-related

risk factor for AD 26. Moreover, p300-medi-

ated autophagy promoted neuronal damage

and inflammation in AD 27. Therefore, p300

may promote the pathogenesis of AD. In this

study, p300 was upregulated in an in vitro

AD model. Targeting p300 may be an effec-

tive strategy for alleviating AD 25,26. In this

study, p300 deficiency induced by a specific

inhibitor suppressed neuroinflammation and

pyroptosis in the neurons. These findings

suggest that p300 inhibition may alleviate

neuronal loss during AD pathogenesis, con-

sistent with previous studies.

p300, an acetyl transferase, has no

DNA-binding domain 28. p300 regulates gene

expression by interacting with transcription

factors. For instance, Sox8 induces the acti-

vation of the LIF interleukin 6 family cyto-

kine downstream transcription factor signal

transducer and activator of transcription 3

via p300 to promote astrocytic differentia-

Fig. 7. p300-depedent activation of NF-κB epigenetically activates NLRP3.

(A) RT-qPCR was conducted to detect mRNA expression in HT-22 cells. (B) A hypothesis of NF-κB/

NLRP3 signalling. (C) JASPAR was used to analyze the binding motif of NF-κB. (D) JASPAR was used

to analyze the binding sites between NF-κB and the promoter of NLRP3. (E) Luciferase assay was

conducted to confirm that the p300/NF-κB promoted the transcription of NLRP3 in HEK293T cells.

(F) Luciferase assay was performed to verify the exact binding site between NF-κB and the promoter

of NLRP3 in HEK293T cells. (G-H) ChIP assay was performed to verify the binding sites in HEK293T

cells. The difference in comparison was analyzed using one-way or two-way ANOVA. p300: E1A binding

protein p300; NLRP3: NLR family pyrin domain containing 3; WT: wild type; MUT: mutant type; RT-

qPCR: reverse transcription-quantitative polymerase chain reaction. ChIP: chromatin immunopreci-

pitation. **p<0.01, ***p<0.001.

Effect of the p300/NF-kB pathway in an in vitro model of Alzheimer’s disease 201

Vol. 66(2): 191 - 204, 2025

tion 29. Moreover, REST recruits CBP/p300

to the EAAT2 promoter to alleviate manga-

nese-induced excitotoxicity 30. Geong et al.

31 reported that p300/NF-κB promotes mi-

croglial activation and neuroinflammation.

In the present study, p300 activated NF-κB.

NF-κB is a key regulator of inflammatory

signalling. NF-κB is a key mediator of brain

inflammation in AD 32. Activating NF-κB sig-

naling stimulates the innate immune system

and induces neurodegeneration and neuro-

nal loss 33-35. In this study, overexpression of

NF-κB antagonized the effects of GNE-049

and promoted neuroinflammation and neu-

ronal death.

Pyroptosis is a type of inflammation-re-

lated cell death process. Inflammation also

induces necroptosis 36. Although pyropto-

sis and necroptosis share some properties,

such as lytic and inflammatory types of pro-

grammed cell death and releasing damage-

associated molecular patterns, pyroptosis

is differentiated from necroptosis, a backup

cell death defense mechanism. In contrast,

pyroptosis is a primary cellular response af-

ter sensing potentially damaging insults37.

Necroptosis is characterized by the activa-

tion of receptor-interacting serine/threonine

kinase 3/mixed lineage kinase domain-like

pseudokinase signalling, whereas pyropto-

sis is characterized by the activation of in-

flammasomes and executed by GSDMD38. In

this study, p300-dependent upregulation of

NF-κB activated the NLRP3 inflammasome

and increased the release of IL-1β and IL-18

after Aβ1-42 exposure. Therefore, the p300/

NF-κB/NLRP3 pathway-mediated neuronal

death occurred via pyroptosis.

In conclusion, p300 was upregulated in

an in vitro model of AD. p300-mediated up-

regulation of NF-κB epigenetically activates

the NLRP3 inflammasome and pyroptosis in

neurons. However, p300 inhibition alleviated

neuroinflammation and neuronal pyroptosis.

Therefore, targeting p300/NF-κB/NLRP3 may

be a promising strategy for alleviating AD.

Ethics approval and consent to participate

This study was approved by the Ethics

Committee of the Third People’s Hospital of

Yunnan Province. All experimental animal

procedures followed the Guidelines for the

Care and Use of Laboratory Animals for-

mulated by China’s Ministry of Science and

Technology.

Funding

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

The datasets used and/or analyzed dur-

ing the current study are available from the

corresponding author upon reasonable re-

quest.

Competing interests

Not applicable.

Number ORCID of authors

• Fengqin Sun (FQS):

0009-0009-0865-8011

• Wei Huang (WH):

0009-0008-9840-1633.

Author contributions

FQS performed data analysis and draft-

ed the manuscript. FQS and WH conceived

and supervised the study and reviewed the

manuscript. FQS and WH ran the software

and modified the code. FQS and WH were

involved in the study design and contributed

to the data collection procedure and inter-

pretation.

ACKNOWLEDGEMENTS

Not applicable.

202 Sun and Huang

Investigación Clínica 66(2): 2025

Conflicts of interest

The authors declared that they have no

conflicts of interest regarding this work.

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