Sinomenine regulates CD14/TLR4, JAK2/STAT3 pathway and calcium signal via α7nAChR to inhibit inflammation in LPS-stimulated macrophages

Abstract

Objective: To investigate the cellular mechanism that sinomenine (SIN) inhibits inflammation in macrophages induced by LPS through α7 nicotinic acetylcholine receptor (α7nAChR).

Materials and methods: RAW264.7 cells were stimulated with LPS and treated by SIN or nicotine (Nic). A selective antagonist of α7nAChR, α-bungarotoxin (BTX) was used to block α7nAChR. AG490 was used to inhibit JAK2 activation. ELISA was performed to detect the levels of TNF-α and MCP-1. Western blotting was used to analyze the expression of MIF, MMP-9, CD14, TLR4, STAT3 and p-STAT3. Intracellular-free calcium level was measured by Fluorescent probe fluo-3/AM

Results: SIN inhibited the production of TNF-α, MCP-1, MIF, and MMP-9, decreased the expression of CD14 and TLR4, and inhibited the release of intracellular-free calcium from intracellular stores in RAW 264.7 cells stimulated by LPS. JAK-specific inhibitor AG490 attenuated the inhibitory effect of SIN on TNF-α. SIN increased the phosphorylation of STAT3. And the above effects of SIN were attenuated by antagonist of α7nAChR.

Conclusions: SIN can decrease the expression of CD14/TLR4 and intracellular free calcium level, activate JAK2/STAT3 pathway to inhibit inflammatory response through α7nAChR in macrophages.

Keywords:

• Sinomenine
• α7 nicotinic acetylcholine receptor
• anti-inflammation
• signaling pathway
• target

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (81573672), the open project of the State Key Laboratory of Quality Research in Chinese Medicine (Macau University of Science and Technology, MUST-SKL-2016–03) funded by the Macao Science and Technology Development Fund, Macau Special Administrative Region.