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Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line
Published on: August 2, 2021
[Paeonol inhibits macrophage M1 polarization by down-regulating miR-155/JAK1-STAT1 pathway]
Ying Sun1, Ling Liu1, Xiao-Yan Shi1
1College of Pharmacy, Anhui University of Chinese Medicine Hefei 230012, China.
Abstract:
The aim of this paper was to investigate the effect and mechanism of paeonol on peritoneal macrophage M1 polarization in mice, explore whether the intervention action is related to the down-regulation of miR-155 and the inhibition of downstream JAK1-STAT1 pathway, and provide a new idea for the molecular mechanism of paeonol against atherosclerosis(AS). Lipopolysaccharide(LPS) and interferon-γ(IFN-γ) were used to stimulate macrophages for 24 hours to establish the M1 polarization model, and paeonol was given 24 hours before co-stimulation to provide a pre-protective effect on cells. CCK-8 assay was used to detect the cells damage induced by LPS and IFN-γ co-stimulation; flow cytometry was used to detect the expression of M1 surface markers F4/80 and CD86. ELISA was used to detect the secretion of interleukin 6(IL-6) and tumor necrosis factor-α(TNF-α) in supernatant. RT-qPCR was used to detect the expression of miR-155, and Western blot was used to detect the protein expression at JAK1-STAT1-SOCS1 pathway. The results showed that LPS and IFN-γ had no obvious damage to the cells at the optimal concentration, but they induced macrophages polarized to M1, resulted in high expression of M1 type marker factors F4/80 and CD86 on the cell surface, and increased secretion of IL-6 and TNF-α on the cell surface(P<0.05 or P<0.01). Paeonol significantly reduced the LPS and IFN-γ-induced high expression of F4/80 and CD86, the secretion of inflammatory factors IL-6 and TNF-α(P<0.05 or P<0.01), decreased the expression level of miR-155, significantly down-regulated the protein phosphorylation level of JAK1-STAT1 and up-regulated the protein expression of SOCS1(P<0.01) in RAW264.7 cells. The results showed that paeonol could inhibit M1 polarization of macrophages by down-regulating cell surface marker factors and inflammatory factors secreted by cells, which may be related to the down-regulation of miR-155 expression and the inhibition JAK1-STAT1 pathway activation.
Insights
Paeonol inhibits M1 polarization in macrophages by down-regulating miR-155 and the JAK1-STAT1 pathway. This finding offers a new therapeutic strategy for atherosclerosis (AS).
Area of Science:
- Immunology
- Pharmacology
- Molecular Biology
Background:
- Macrophage polarization, particularly M1 polarization, plays a critical role in the pathogenesis of atherosclerosis (AS).
- Identifying novel therapeutic agents that can modulate macrophage polarization is crucial for AS treatment.
Purpose of the Study:
- To investigate the effect of paeonol on M1 polarization of peritoneal macrophages in mice.
- To elucidate the underlying molecular mechanism involving miR-155 and the JAK1-STAT1 pathway.
- To explore paeonol as a potential therapeutic strategy for atherosclerosis.
Main Methods:
- Establishment of M1 macrophage polarization model using lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) stimulation.
- Assessment of paeonol's protective effects using CCK-8 assay, flow cytometry (F4/80, CD86), and ELISA (IL-6, TNF-α).
- Analysis of miR-155 expression via RT-qPCR and JAK1-STAT1-SOCS1 pathway protein levels using Western blot.
Main Results:
- LPS and IFN-γ successfully induced M1 polarization, evidenced by increased F4/80, CD86, IL-6, and TNF-α.
- Paeonol significantly attenuated M1 polarization markers and inflammatory cytokine secretion.
- Paeonol treatment led to decreased miR-155 expression, reduced JAK1-STAT1 phosphorylation, and increased SOCS1 protein levels.
Conclusions:
- Paeonol effectively inhibits M1 macrophage polarization.
- The mechanism involves down-regulation of miR-155 and inhibition of the JAK1-STAT1 pathway.
- Paeonol demonstrates potential as a novel therapeutic agent for atherosclerosis.
