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MiR-146a-5p Contributes to Microglial Polarization Transitions Associated With AGEs

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Abstract

M1/M2 polarization transitions of microglial phenotypes determine the states of neuroinflammation, which is critical in the pathophysiology of diabetic encephalopathy. This study aims to investigate the effects of advanced glycation end products (AGEs) on the microglial polarization state, the role of miR-146a-5p in the regulation of microglial polarization, and the underlying signaling pathways. BV-2 cells were incubated with N-ε-carboxymethyl lysine (CML), one kind of Advanced Glycation End Products (AGEs), to induce polarization. CD11b and iNOS and CD206 and Arg-1 were used to evaluate M1 and M2 microglia, respectively. The mRNA and protein expression levels of miR-146a-5p, transcription factor NF-κB, and inflammasome NLRP3 were measured. High and low expression of miR-146a-5p in the BV-2 cell line was generated by lentivirus transfection technology. RAGE, TLR-4, and NF-κB antagonists were applied to evaluate the underlying signaling pathways. Compared with the control group, CML upregulated the M1 phenotype and downregulated the M2 phenotype. These effects were reversed by overexpression of miR-146a. Furthermore, the expression of inflammasome NLRP3 and NF-κB was upregulated in the CML group and was reduced after miR-146a overexpression. And then overexpression of miR-146a effects was reversed by inhibition miR-146a expression. An NF-κB antagonist (PDTC), a RAGE antagonist (FPS-ZMI), and a TLR-4 antagonist (TLI-095) all reversed the polarization state induced by CML. In summary, CML induced polarization transitions to M1 phenotype and promoted inflammasome NLRP3 expression in BV-2 cells. The RAGE or TLR-4/miR-146a/NLRP3/NF-кB pathway might participate in the regulation of CML-induced BV-2 polarization.

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Data Availability

All relevant data is contained within the article. Further inquiries can be directed to the corresponding author.

Abbreviations

APS:

Ammonium persulfate

BSA:

Albumin from bovine serum

BCA:

Bicinchoninic acid

BV-2:

BV-2 microglia

CML:

Nε-carboxymethyl-lysine

CAS:

Chinese Academy of Sciences

CDNA:

Complementary DNA

DEPC:

Diethyl pyrocarbonate

DMSO:

Dimethyl sulfoxide

ECL:

Enhanced chemiluminescence

FBS:

Fetal bovine serum

GFP:

Green fluorescence protein

MOI:

Multiplicity of infection

PCR:

Polymerase chain reaction

PAGE:

Polyacrylamide gel electrophoresis

PBS:

Phosphate-buffered solution

PMSF:

Phenylmethanesulfonyl fluoride

PVDF:

Polyvinylidene fluoride

RPM:

Revolutions per minute

RPMI:

RPMI Medium 1640 basic (1 ×)

RAGE:

Receptors of advanced glycation end products

SDS:

Sodium dodecyl sulfate

TBS:

Tris-buffered saline

TEMED:

Tetraaminoethylenediamine

TLR-4:

Toll-like receptor-4

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Funding

Funding for this work was supported by the National Natural Science Foundation of China (NSFC No.82200871,82270873), Natural Science Foundation of Fujian Province (2020J01221, 2020J01226), Medical Innovation Project of Fujian Province (2020CXA044), Science Fund for Distinguished Young Scholars of Fujian Province (2020GGA057), and the Postdoctoral Start-up fund of the Second Affiliated Hospital of Fujian Medical University (2020BSH02).

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  1. Yinqiong Huang and Xiaoyun Lin contributed equally to this manuscript.

Authors and Affiliations

  1. Department of Endocrinology, Centre of Neurological and Metabolic Research, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian Province, 360000, China

    Yinqiong Huang

  2. Department of Clinical Nutrition, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, Fujian, China

    Xiaoyun Lin

  3. Department of Endocrinology, Department of Geriatrics, The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen, 518000, Guangdong, China

    Xiahong Lin

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  1. Yinqiong Huang
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Contributions

Yinqiong Huang and Xiaoyun Lin contributed equally to this manuscript. Yinqiong Huang, Xiaoyun Lin, and Xiahong Lin conceptualized and designed these studies, performed them, and wrote the manuscript. All authors contributed to manuscript revision and read and approved the submitted version.

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Correspondence to Yinqiong Huang or Xiahong Lin.

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Huang, Y., Lin, X. & Lin, X. MiR-146a-5p Contributes to Microglial Polarization Transitions Associated With AGEs. Mol Neurobiol 60, 3020–3033 (2023). https://doi.org/10.1007/s12035-023-03252-8

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