Sinomenine regulates CD14/TLR4, JAK2/STAT3 pathway and calcium signal via 7nAChR to inhibit inflammation in LPS-stimulated macrophages

Objective: To investigate the cellular mechanism that sinomenine (SIN) inhibits inflammation in macrophages induced by LPS through 7 nicotinic acetylcholine receptor (7nAChR).Materials and methods: RAW264.7 cells were stimulated with LPS and treated by SIN or nicotine (Nic). A selective antagonist of 7nAChR, -bungarotoxin (BTX) was used to block 7nAChR. AG490 was used to inhibit JAK2 activation. ELISA was performed to detect the levels of TNF- 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/AMResults: SIN inhibited the production of TNF-, 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-. SIN increased the phosphorylation of STAT3. And the above effects of SIN were attenuated by antagonist of 7nAChR.Conclusions: SIN can decrease the expression of CD14/TLR4 and intracellular free calcium level, activate JAK2/STAT3 pathway to inhibit inflammatory response through 7nAChR in macrophages.