Institute of Digestive Disease, China Three Gorges University, Yichang, 443000, China.
Department of Gastroenterology, Yichang Central People's Hospital, Yichang, 443000, China.
Curr Med Sci. 2020 Feb;40(1):130-137. doi: 10.1007/s11596-020-2156-6. Epub 2020 Mar 13.
Sinomenine (SN) has been used in the clinical treatment of systemic lupus erythematosus and rheumatoid arthritis for many years. Studies showed that SN held protective effects such as anti-inflammation, scavenging free radicals and suppressing immune response in many autoimmune diseases. The purpose of the present study is to explore the mechanism of anti-inflammation of SN on lipopolysaccharide (LPS)-induced macrophages activation and investigate whether the TLR4/NF-κB signaling pathway participated in. Macrophages isolated from mouse peritoneal cavity were stimulated by 1 µg/mL LPS for 24 h. And then the cells were treated with various concentrations of SN, TLR4 inhibitor respectively for additional 48 h. Drug toxicity was detected by MTT assay and Transwell experiment was used to assess chemotaxis. Furthermore, TLR4 and MyD88 mRNA levels were detected by real-time PCR. Western blotting was used to examine TLR4, MyD88 and phosphorylated IκB protein expression in macrophages. Immunofluorescence assay was applied to observe p65 NF-κB protein expression in macrophage nucleus. We extracted macrophages with high purity and activity from the abdominal cavity of mice. SN remarkably inhibited the chemotaxis and secretion function of LPS-stimulated macrophages. It also down-regulated both the protein levels of inflammatory cytokines (TNF-α, IL-1β and IL-6) and the RNA and protein levels of the key factors (TLR4, MyD88, P-IκB) in TLR4 pathway. The expression of p65 NF-κB protein in nuclei was down-regulated, which was correlated with a similar decrease in P-IκB protein level. In conclusion, SN can inhibit the LPS induced immune responses in macrophages by blocking the activated TLR4/NF-κB signaling pathway. These results may provide a therapeutic approach to regulate inflammatory responses.
青藤碱 (SN) 多年来一直用于系统性红斑狼疮和类风湿关节炎的临床治疗。研究表明，SN 在许多自身免疫性疾病中具有抗炎、清除自由基和抑制免疫反应等保护作用。本研究旨在探讨 SN 对脂多糖 (LPS) 诱导的巨噬细胞活化的抗炎作用机制，并研究 TLR4/NF-κB 信号通路是否参与其中。用 1μg/mL LPS 刺激小鼠腹腔分离的巨噬细胞 24 h。然后用不同浓度的 SN 和 TLR4 抑制剂分别处理细胞 48 h。MTT 法检测药物毒性，Transwell 实验检测趋化作用。进一步通过实时 PCR 检测 TLR4 和 MyD88 mRNA 水平。用 Western blot 检测巨噬细胞中 TLR4、MyD88 和磷酸化 IκB 蛋白的表达。免疫荧光法观察巨噬细胞核中 p65 NF-κB 蛋白的表达。我们从小鼠腹腔中提取出高纯度和高活性的巨噬细胞。SN 显著抑制 LPS 刺激的巨噬细胞的趋化作用和分泌功能。它还下调了 TLR4 通路中关键因子（TLR4、MyD88、P-IκB）的蛋白水平和 RNA 及蛋白水平的炎症细胞因子（TNF-α、IL-1β 和 IL-6）。核内 p65 NF-κB 蛋白的表达下调，与 P-IκB 蛋白水平的相似下降相关。综上所述，SN 通过阻断激活的 TLR4/NF-κB 信号通路，抑制 LPS 诱导的巨噬细胞免疫反应。这些结果可能为调节炎症反应提供一种治疗方法。
