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RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization
BACKGROUND: DNA methyltransferase 1 (DNMT1) exerts imperative functions in neuropathic pain (NP). This study explored the action of RNA interference-mediated DNMT1 silencing in NP by regulating microglial M2 polarization. METHODS: NP rat models were established using chronic constriction injury (CCI...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9526327/ https://www.ncbi.nlm.nih.gov/pubmed/36183073 http://dx.doi.org/10.1186/s12883-022-02860-6 |
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author | Tan, Ying Wang, Zongjiang Liu, Tao Gao, Peng Xu, Shitao Tan, Lei |
author_facet | Tan, Ying Wang, Zongjiang Liu, Tao Gao, Peng Xu, Shitao Tan, Lei |
author_sort | Tan, Ying |
collection | PubMed |
description | BACKGROUND: DNA methyltransferase 1 (DNMT1) exerts imperative functions in neuropathic pain (NP). This study explored the action of RNA interference-mediated DNMT1 silencing in NP by regulating microglial M2 polarization. METHODS: NP rat models were established using chronic constriction injury (CCI) and highly aggressive proliferating immortalized (HAPI) microglia were treated with lipopolysaccharide (LPS) to induce microglia M1 polarization, followed by treatment of DNMT1 siRNA or si-DNMT1/oe-DNMT1, respectively. The pain threshold of CCI rats was assessed by determining mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL). Levels of inflammatory factors (TNF-α/IL-1β/IL-6/IL-10) and DNMT1 in rat L4-L6 spinal cord samples and HAPI cells were measured using ELISA, RT-qPCR, and Western blot. iNOS and Arg-1 mRNA levels were measured via RT-qPCR. DNMT1, M1 marker (iNOS), and M2 marker (Arg-1) levels in microglia of CCI rats were detected by immunofluorescence. Percentages of M1 microglia phenotype (CD16) and M2 microglia phenotype (CD206) were detected by flow cytometry. The phosphorylation of PI3K/Akt pathway-related proteins was determined by Western blot. RESULTS: CCI rats exhibited diminished MWT and TWL values, increased pro-inflammatory cytokines, and decreased anti-inflammatory cytokine IL-10. Additionally, DNMT1 was upregulated in CCI rat microglia. DNMT1 siRNA alleviated CCI-induced NP and facilitated M2 polarization of microglia in CCI rats. DNMT1 knockdown inhibited LPS-induced M1 polarization of HAPI cells and promoted M2 polarization by blocking the PI3K/Akt pathway, but DNMT1 overexpression inhibited the M1-to-M2 polarization of microglia. CONCLUSION: RNA interference-mediated DNMT1 silencing accelerates microglia M2 polarization by impeding the PI3K/Akt pathway, thereby alleviating CCI-induced NP. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12883-022-02860-6. |
format | Online Article Text |
id | pubmed-9526327 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-95263272022-10-02 RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization Tan, Ying Wang, Zongjiang Liu, Tao Gao, Peng Xu, Shitao Tan, Lei BMC Neurol Research BACKGROUND: DNA methyltransferase 1 (DNMT1) exerts imperative functions in neuropathic pain (NP). This study explored the action of RNA interference-mediated DNMT1 silencing in NP by regulating microglial M2 polarization. METHODS: NP rat models were established using chronic constriction injury (CCI) and highly aggressive proliferating immortalized (HAPI) microglia were treated with lipopolysaccharide (LPS) to induce microglia M1 polarization, followed by treatment of DNMT1 siRNA or si-DNMT1/oe-DNMT1, respectively. The pain threshold of CCI rats was assessed by determining mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL). Levels of inflammatory factors (TNF-α/IL-1β/IL-6/IL-10) and DNMT1 in rat L4-L6 spinal cord samples and HAPI cells were measured using ELISA, RT-qPCR, and Western blot. iNOS and Arg-1 mRNA levels were measured via RT-qPCR. DNMT1, M1 marker (iNOS), and M2 marker (Arg-1) levels in microglia of CCI rats were detected by immunofluorescence. Percentages of M1 microglia phenotype (CD16) and M2 microglia phenotype (CD206) were detected by flow cytometry. The phosphorylation of PI3K/Akt pathway-related proteins was determined by Western blot. RESULTS: CCI rats exhibited diminished MWT and TWL values, increased pro-inflammatory cytokines, and decreased anti-inflammatory cytokine IL-10. Additionally, DNMT1 was upregulated in CCI rat microglia. DNMT1 siRNA alleviated CCI-induced NP and facilitated M2 polarization of microglia in CCI rats. DNMT1 knockdown inhibited LPS-induced M1 polarization of HAPI cells and promoted M2 polarization by blocking the PI3K/Akt pathway, but DNMT1 overexpression inhibited the M1-to-M2 polarization of microglia. CONCLUSION: RNA interference-mediated DNMT1 silencing accelerates microglia M2 polarization by impeding the PI3K/Akt pathway, thereby alleviating CCI-induced NP. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12883-022-02860-6. BioMed Central 2022-10-01 /pmc/articles/PMC9526327/ /pubmed/36183073 http://dx.doi.org/10.1186/s12883-022-02860-6 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. 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/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Tan, Ying Wang, Zongjiang Liu, Tao Gao, Peng Xu, Shitao Tan, Lei RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization |
title | RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization |
title_full | RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization |
title_fullStr | RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization |
title_full_unstemmed | RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization |
title_short | RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization |
title_sort | rna interference-mediated silencing of dna methyltransferase 1 attenuates neuropathic pain by accelerating microglia m2 polarization |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9526327/ https://www.ncbi.nlm.nih.gov/pubmed/36183073 http://dx.doi.org/10.1186/s12883-022-02860-6 |
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