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Article Open Access

miR‑223‑3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis

  • Authors:
    • Xiaoyu Wang
    • Lin Song
    • Jiafeng Wang
    • Qianqian Xie
    • Yan Wang
    • Chunyan Li
    • Tianqi Wang
    • Yifeng Du
  • View Affiliations / Copyright

    Affiliations: Department of Neurology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250021, P.R. China, Department of Urology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250021, P.R. China
    Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 178
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    Published online on: May 8, 2026
       https://doi.org/10.3892/ijmm.2026.5849
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Abstract

Neuroinflammation is a hallmark of Alzheimer's disease (AD) and is closely linked to microglial M1 polarization. In the present study, miR‑223‑3p was identified as a critical regulator of microglial metabolic reprogramming. Analyses of Gene Expression Omnibus and AD Neuroimaging Initiative datasets revealed significant upregulation of miR‑223‑3p in the brain, blood, and cerebrospinal fluid of patients with AD. The overexpression of miR‑223‑3p promoted M1 polarization and increased reactive oxygen species (ROS) levels. Transcriptomic, metabolomic and Seahorse analyses revealed increased glycolysis, lactate production and lactylation, whereas inhibition of lactylation reduced M1 polarization and ROS accumulation. Mechanistically, miR‑223‑3p suppressed SIRT1 expression and directly targeted FBXW7, leading to activation of the Notch1/Hes1 pathway and further suppression of SIRT1. In summary, these findings demonstrate that miR‑223‑3p drives microglial lactylation‑mediated M1 polarization through the FBXW7/Notch1/Hes1/SIRT1 signaling axis. The present study provides new insight into the role of lactylation in neuroinflammation and highlights miR‑223‑3p as a potential therapeutic target for AD.
View Figures

Figure 1

miR-223-3p is dysregulated in
patients with AD. (A and B) Expression of miR-223-3p in (A)
parietal lobe tissues and (B) prefrontal cortex tissues of patients
with AD compared with controls. (C and D) miR-223-3p levels in both
(C) serum and (D) CSF samples from patients with AD relative to
those from controls in the GEO dataset. (E and F) miR-223-3p levels
in both (E) serum and (F) CSF samples from patients with AD
relative to those from controls according to the ADNI dataset. (G)
ROC curve of miR-223-3p in parietal lobe tissues for AD from GEO
dataset. (H) ROC curve of miR-223-3p in prefrontal cortex tissues
for AD from GEO dataset. (I) ROC curves of miR-223-3p in serum for
AD from GEO dataset. (J) ROC curves of miR-223-3p in CSF for AD
from GEO dataset. (K) ROC curves of miR-223-3p in serum for AD from
ADNI dataset. (L) ROC curves of miR-223-3p in CSF for AD from ADNI
dataset. *P<0.05, **P<0.01 and
***P<0.001. miR, microRNA; AD, Alzheimer's disease;
CSF, cerebrospinal fluid; GEO, Gene Expression Omnibus; ADNI, AD
Neuroimaging Initiative; ROC, receiver operating characteristic;
AUC, area under the curve; CI, confidence interval.

Figure 2

miR-223-3p promotes microglial M1
polarization. (A) RT-qPCR analysis of miR-223-3p expression. (B)
RT-qPCR analysis of M1/M2 polarization marker genes in microglia.
(C) Western blot detection of iNOS and Arg-1 protein levels. (D)
Flow cytometric analysis of ROS levels. (E) Heatmap of genes
differentially expressed between the high- and low-miR-223-3p
groups. (F) KEGG pathway enrichment analysis of differentially
expressed genes. (G) Gene Set Enrichment Analysis of
glycolysis-associated pathways in miR-223-3p-regulated microglia.
**P<0.01 and ***P<0.001. miR, microRNA;
RT-qPCR, reverse transcription-quantitative PCR; iNOS, inducible
nitric oxide synthase; ROS, reactive oxygen species; KEGG, Kyoto
Encyclopedia of Genes and Genomes; NC, negative control.

Figure 3

miR-223-3p promotes microglial M1
polarization through the regulation of lactylation. (A) Heatmap of
metabolite profiles across three experimental groups. (B-E)
LC-MS/MS quantification of (B) lactate, (C) pyruvate, (D)
NADP+ and (E) NADPH levels. (F and G) ECAR assays
measuring basal and compensatory glycolysis. (H and I) OCR assays
assessing mitochondrial respiration. (J) Lactate content assay
assessing intracellular lactate level. (K) Western blot analysis of
global lactylation levels. (L) Western blot analysis of lactylation
levels following treatment with glycolytic inhibitors oxamate (10
mM; 24 h) and 2-DG (10 mM; 24 h). (M) Reverse
transcription-quantitative PCR analysis of microglial polarization
markers. (N and O) Flow cytometric analysis of ROS levels.
*P<0.05, **P<0.01 and
***P<0.001. miR, microRNA; ECAR, extracellular
acidification rate; OCR, oxygen consumption rate; 2-DG,
2-deoxy-D-glucose; ROS, reactive oxygen species; NC, negative
control.

Figure 4

miR-223-3p promotes microglial
lactylation and M1 polarization by suppressing SIRT1 expression.
(A) RT-qPCR analysis of SIRT1 mRNA expression. (B) Western
blot analysis of SIRT1 protein levels. (C) Western blot analysis of
lactylation levels following treatment with SIRT1 activator SRT1720
(5 μM; 24 h). (D) Western blot analysis of lactylation
levels following treatment with SIRT1 inhibitor EX-527 (10
μM; 24 h). (E and F) RT-qPCR and Western blot validation of
SIRT1 overexpression in rescue experiments. (G) Western blot
detection of global lactylation levels. (H) RT-qPCR analysis of
microglial polarization markers. (I) Western blot analysis of iNOS
and Arg-1 protein expression. (J and K) Flow cytometric analysis of
ROS levels. *P<0.05, **P<0.01 and
***P<0.001. miR, microRNA; SIRT1, sirtuin 1; RT-qPCR,
reverse transcription-quantitative PCR; iNOS, inducible nitric
oxide synthase; ROS, reactive oxygen species; NC, negative control;
OE, overexpression.

Figure 5

miR-223-3p suppresses SIRT1
transcription via the Notch1/Hes1 signaling pathway, thereby
promoting microglial lactylation and M1 polarization. (A) RT-qPCR
analysis of Notch1 and Hes1 mRNA levels. (B) Western
blot analysis of NICD1 and Hes1 protein expression. (C) Western
blot analysis of SIRT1 expression following silencing Hes1. (D)
Western blot detection of global lactylation levels. (E) RT-qPCR
analysis of microglial polarization markers. (F and G) RT-qPCR and
western blot detection of SIRT1 following treatment with Notch1
inhibitor DAPT (20 nM; 24 h). (H) Western blot detection of global
lactylation levels. (I) RT-qPCR analysis of microglial polarization
markers. (J) Western blot analysis of iNOS and Arg-1 protein
expression. (K) Flow cytometric analysis of ROS levels.
*P<0.05, **P<0.01 and
***P<0.001. miR, microRNA; SIRT1, sirtuin 1; RT-qPCR,
reverse transcription-quantitative PCR; iNOS, inducible nitric
oxide synthase; ROS, reactive oxygen species; NC, negative control;
ns, not significant.

Figure 6

miR-223-3p targets FBXW7 to reduce
Notch1 ubiquitination and degradation. (A) Venn diagram showing the
overlap of predicted miR-223-3p targets from three databases. (B)
Reverse transcription-quantitative PCR analysis of downstream
target mRNA expression. (C) Western blot detection of FBXW7 protein
expression. (D) Predicted binding site between miR-223-3p and the
3' untranslated region of FBXW7. (E) Dual-luciferase
reporter assay confirming the direct interaction between miR-223-3p
and FBXW7. (F) Effects of the proteasome inhibitor MG132 and
the lysosomal inhibitor CQ on NICD1 protein expression in BV2 cells
with altered miR-223-3p expression. (G) CHX chase assay showing the
dynamics of NICD1 protein degradation in BV2 cells. (H and I)
Co-immunoprecipitation analysis of the interaction between FBXW7
and NICD1. (J) Effect of FBXW7 overexpression on NICD1
ubiquitination in 293T cells. (K) Western blot analysis of
FBXW7-mediated regulation of NICD1 expression and ubiquitination in
microglia. **P<0.01 and ***P<0.001.
miR, microRNA; CHX, cycloheximide; NC, negative control; ns, not
significant.

Figure 7

Schematic representation of the
proposed mechanism of miR-223-3p in microglial M1 polarization.
miR, microRNA; SIRT1, sirtuin 1.
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Copy and paste a formatted citation
Spandidos Publications style
Wang X, Song L, Wang J, Xie Q, Wang Y, Li C, Wang T and Du Y: miR‑223‑3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis. Int J Mol Med 58: 178, 2026.
APA
Wang, X., Song, L., Wang, J., Xie, Q., Wang, Y., Li, C. ... Du, Y. (2026). miR‑223‑3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis. International Journal of Molecular Medicine, 58, 178. https://doi.org/10.3892/ijmm.2026.5849
MLA
Wang, X., Song, L., Wang, J., Xie, Q., Wang, Y., Li, C., Wang, T., Du, Y."miR‑223‑3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis". International Journal of Molecular Medicine 58.1 (2026): 178.
Chicago
Wang, X., Song, L., Wang, J., Xie, Q., Wang, Y., Li, C., Wang, T., Du, Y."miR‑223‑3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis". International Journal of Molecular Medicine 58, no. 1 (2026): 178. https://doi.org/10.3892/ijmm.2026.5849
Copy and paste a formatted citation
x
Spandidos Publications style
Wang X, Song L, Wang J, Xie Q, Wang Y, Li C, Wang T and Du Y: miR‑223‑3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis. Int J Mol Med 58: 178, 2026.
APA
Wang, X., Song, L., Wang, J., Xie, Q., Wang, Y., Li, C. ... Du, Y. (2026). miR‑223‑3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis. International Journal of Molecular Medicine, 58, 178. https://doi.org/10.3892/ijmm.2026.5849
MLA
Wang, X., Song, L., Wang, J., Xie, Q., Wang, Y., Li, C., Wang, T., Du, Y."miR‑223‑3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis". International Journal of Molecular Medicine 58.1 (2026): 178.
Chicago
Wang, X., Song, L., Wang, J., Xie, Q., Wang, Y., Li, C., Wang, T., Du, Y."miR‑223‑3p promotes microglial lactylation and M1 polarization via the FBXW7/Notch1/Hes1/SIRT1 axis". International Journal of Molecular Medicine 58, no. 1 (2026): 178. https://doi.org/10.3892/ijmm.2026.5849
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