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RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia
Ying Tan1, Zongjiang Wang2, Tao Liu3
1Department of Spinal Surgery, Weifang Traditional Chinese Medicine Hospital, No.1055, Weizhou Road, Kuiwen District, Weifang, 261041, China. tanying335@163.com.
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.
Insights
Silencing DNA methyltransferase 1 (DNMT1) using RNA interference promotes M2 microglia polarization and alleviates neuropathic pain (NP) by inhibiting the PI3K/Akt pathway.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- DNA methyltransferase 1 (DNMT1) plays a crucial role in neuropathic pain (NP).
- Microglial polarization (M1/M2 phenotypes) is critical in NP pathogenesis.
- This study investigates DNMT1's role in regulating microglial polarization in NP.
Purpose of the Study:
- To explore the effect of RNA interference-mediated DNMT1 silencing on neuropathic pain.
- To investigate the mechanism by which DNMT1 affects microglial M2 polarization.
- To determine the involvement of the PI3K/Akt pathway in DNMT1-mediated microglial modulation.
Main Methods:
- Neuropathic pain rat models were induced by chronic constriction injury (CCI).
- Microglial polarization was manipulated using DNMT1 siRNA and overexpression in vitro and in vivo.
- Pain thresholds, inflammatory cytokine levels, and PI3K/Akt pathway activation were assessed.
Main Results:
- CCI rats showed increased DNMT1 expression, pro-inflammatory cytokines, and NP behaviors.
- DNMT1 silencing alleviated NP symptoms and promoted M2 microglia polarization in CCI rats.
- DNMT1 knockdown inhibited M1 polarization and promoted M2 polarization by blocking the PI3K/Akt pathway.
Conclusions:
- RNA interference-mediated DNMT1 silencing accelerates microglia M2 polarization.
- Inhibition of the PI3K/Akt pathway is a key mechanism in DNMT1-silencing-induced M2 polarization.
- DNMT1 silencing demonstrates therapeutic potential for alleviating neuropathic pain.
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