Sinomenine inhibits macrophage M1 polarization by downregulating α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1

Background: Sinomenine (SIN) is an anti-inflammatory drug that has been used for decades in China to treat arthritis. In a previous study, SIN acted on alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR) to inhibit inflammatory responses in macrophages, which indicates a new anti-inflammatory mechanism of SIN. However, the level of alpha 7nAChR was increased in the inflammatory responses and was downregulated by SIN in vitro, so the underlying mechanisms of SIN acting on alpha 7nAChR remain unclear. Purpose: To analyze the role of alpha 7nAChR in inflammation and the effect and mechanism of SIN regulation of alpha 7nAChR. Methods: The effects of SIN on alpha 7nAChR in endotoxemic mice and LPS-stimulated macrophages were observed. Nicotine (Nic) was used as a positive control, and berberine (Ber) was used as a negative control targeting alpha 7nAChR. The antagonists of alpha 7nAChR, alpha-bungarotoxin (BTX) and mecamylamine (Me), were used to block alpha 7nAChR. In RAW264.7 macrophage cells in vitro, alpha 7nAChR short hairpin RNA (shRNA) was used to knock down alpha 7nAChR. Macrophage polarization was analyzed by the detection of TNF-alpha, IL-6, iNOS, IL-10, Arg-1, and Fizz1. U0126 was used to block ERK phosphorylation. The cytokines alpha 7nAChR, ERK1/2, p-ERK1/2 and Egr-1 were detected. Results: SIN decreased the levels of TNF-alpha, IL-6 and the expression of alpha 7nAChR increased by LPS in endotoxemic mice. The above effects of SIN were attenuated by BTX. In the alpha 7nAChR shRNA transfected RAW264.7 cells, compared with the control, alpha 7nAChR was knocked down, and M1 phenotype markers (including TNF-alpha, IL-6, and iNOS) were significantly downregulated, whereas M2 phenotype markers (including IL-10, Arg-1, and Fizz1) were significantly upregulated when stimulated by LPS. SIN inhibited the expression of p-ERK1/2 and the transcription factor Egr-1 induced by LPS in RAW264.7 cells, and the above effects of SIN were attenuated by BTX. The expression of alpha 7nAChR was suppressed by U0126, which lessened the expression of p-ERK1/2 and Egr-1. Conclusions: SIN acts on alpha 7nAChR to inhibit inflammatory responses and downregulates high expression of alpha 7nAChR in vivo and in vitro. The increase of alpha 7nAChR expression is correlated with inflammatory responses and participates in macrophage M1 polarization. SIN downregulates alpha 7nAChR via a feedback pathway of alpha 7nAChR/ERK/Egr-1, which contributes to inhibiting macrophage M1 polarization and inflammatory responses.

Automated summary

The study found that Sinomenine (SIN) inhibits inflammatory responses by acting on alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR) and reduces the high expression of alpha 7nAChR in both in vivo and in vitro settings. The increased expression of alpha 7nAChR is associated with inflammatory responses and is involved in macrophage polarization. SIN downregulates alpha 7nAChR through a feedback pathway of alpha 7nAChR/ERK/Egr-1, which contributes to the inhibition of macrophage M1 polarization and inflammatory responses.