黄精水提物干预脂多糖诱导的巨噬细胞极化与自噬研究

Effect of Polygonatum sibiricum aqueous extract on lipopolysaccharides induced macrophage polarization and autophagy

中文关键词:  黄精水提物  巨噬细胞  极化  炎症  自噬

英文关键词:Polygonatum sibiricum aqueous extract  macrophage  polarization  inflammation  autophagy

中文摘要:

      目的 探讨黄精水提物(Polygonatum sibiricum aqueous extract, PSAE)对脂多糖(lipopolysaccharides, LPS)诱导的RAW264.7巨噬细胞M1极化与自噬的调节作用, 探究其抗炎机制。方法 实验分为对照组(无任何处理)、LPS组(100 ng/mL LPS刺激12 h)、PSAE低、中、高剂量组(50、100、200 μg/mL PSAE预处理细胞 4 h后, 再加入100 ng/mL LPS刺激12 h)。细胞计数(cell counting kit-8, CCK-8)法检测细胞活性; Griess法检测细胞NO分泌量; 实时荧光定量PCR检测细胞诱导型NO合酶(inducible nitric oxide synthase, iNOS)、白细胞介素-1β (interleukin-1β, IL-1β)、肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)及单核细胞趋化蛋白-1 (monocyte chemotactic protein-1, MCP-1)的mRNA水平; 蛋白质免疫印迹和免疫荧光法检测M1型极化标志物iNOS表达水平; 蛋白质免疫印迹检测微管相关蛋白1轻链3 (microtubule-associated protein 1 light chain 3, LC3)和螯合体1 (sequestosome 1, p62)表达水平。结果 与LPS组相比, PSAE可使RAW264.7细胞NO的分泌减少(P<0.05, P<0.01), PSAE也可降低M1极化相关炎症因子(iNOS、IL-1β、TNF-α、MCP-1) mRNA表达水平(P<0.05, P<0.01, P<0.001), 降低M1极化标志物iNOS蛋白表达水平(P<0.05, P<0.001), 同时, PSAE可降低LC3II/I值(P<0.01, P<0.001), 促进p62蛋白表达(P<0.01), 从而降低自噬水平。结论 PSAE能够抑制LPS诱导的RAW264.7细胞M1极化和自噬水平, 减轻细胞炎症反应。

英文摘要:

      Objective To investigate the regulatory effect of Polygonatum sibiricum aqueous extract (PSAE) on the M1 polarization and autophagy of RAW264.7 cells induced by lipopolysaccharides (LPS), and explore its anti-inflammatory effects. Methods The experiment was divided into control group (without any treatment), LPS group (treated with 100 ng/mL LPS for 12 h), PSAE low-dose, medium-dose and high-dose groups (50, 100, 200 μg/mL PSAE pre-treated for 4 h , respectively, and then treated with 100 ng/mL LPS for 12 h). Cell counting kit-8 (CCK-8) assay was used to detect the viability of RAW264.7 cells; Griess assay was used to detect the products of NO; real-time fluorescent quantitative polymerase chain reaction method was used to detect the mRNA expression levels of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and monocyte chemotactic protein (MCP-1); western blot and immunocellular fluorescence assay were used to detect the expression levels of M1 polarization maker, iNOS; the protein expression levels of microtubule-associated protein light chain 3 (LC3) and sequestosome 1 (p62) were detected by western blot. Results Compared with LPS group, PSAE could reduce the secretions of NO (P<0.05, P<0.01), decrease the mRNA expression level of iNOS, IL-1β, TNF-α, and MCP-1 (P<0.05, P<0.01, P<0.001), and decrease the protein expression levels of iNOS (P<0.05, P<0.001), besides, PSAE could reduce the values of LC3II/I (P<0.01, P<0.001), and promote the protein expression of p62 (P<0.01), thus reducing the autophagy levels. Conclusion PSAE could inhibit macrophage M1 polarization and autophagy levels of LPS-stimulated RAW264.7 cells, and alleviate cellular inflammation.