a Department of Immunology, Institute of Clinical Pharmacology , Guangzhou University of Chinese Medicine , Guangzhou , P.R. China.
b Faculty of Chinese Medicine , Macau University of Science and Technology, the State Key Laboratory of Quality Research in Chinese Medicine (Macau University of Science and Technology) , Taipa , P.R. China.
Immunopharmacol Immunotoxicol. 2019 Feb;41(1):172-177. doi: 10.1080/08923973.2019.1568451. Epub 2019 Mar 21.
To investigate the cellular mechanism that sinomenine (SIN) inhibits inflammation in macrophages induced by LPS through α7 nicotinic acetylcholine receptor (α7nAChR). 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 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. 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.
目的：通过α7 烟碱型乙酰胆碱受体（α7nAChR）研究盐酸青藤碱（SIN）抑制脂多糖（LPS）诱导的巨噬细胞炎症的细胞机制。
方法：用 LPS 刺激 RAW264.7 细胞，并用 SIN 或尼古丁（Nic）处理。用α7nAChR 的选择性拮抗剂α-银环蛇毒素（BTX）阻断α7nAChR。用 JAK2 激活抑制剂 AG490 抑制 JAK2 激活。用 ELISA 检测 TNF-α 和 MCP-1 的水平。用 Western blot 分析 MIF、MMP-9、CD14、TLR4、STAT3 和 p-STAT3 的表达。用荧光探针 fluo-3/AM 测量细胞内游离钙水平。
结果：SIN 抑制 LPS 刺激的 RAW264.7 细胞中 TNF-α、MCP-1、MIF 和 MMP-9 的产生，降低 CD14 和 TLR4 的表达，抑制细胞内钙库中细胞内游离钙的释放。JAK 特异性抑制剂 AG490 减弱了 SIN 对 TNF-α 的抑制作用。SIN 增加了 STAT3 的磷酸化。α7nAChR 的拮抗剂减弱了 SIN 的上述作用。
结论：SIN 可通过α7nAChR 降低巨噬细胞中 CD14/TLR4 的表达和细胞内游离钙水平，激活 JAK2/STAT3 通路，抑制炎症反应。
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