College of Traditional Chinese Veterinary Medicine, Agricultural University of Hebei , Baoding, China .
J Interferon Cytokine Res. 2018 Jan;38(1):29-37. doi: 10.1089/jir.2017.0087.
This study investigated the anti-inflammatory effects and possible underlying mechanisms of Salvia miltiorrhiza polysaccharides (SMP) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The cytotoxicity of SMP was detected by the MTT method. The morphological change of RAW264.7 was observed by Diff-Quik staining. Enzyme-linked immunosorbent assay was used to evaluate the production of cytokines in LPS-induced RAW264.7 cells. The nitric oxide (NO) kit assay detected the NO release from LPS-induced RAW264.7 cells. Real-time polymerase chain reaction was used to detect the transcriptions of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), inducible NO synthase (iNOS), and cyclooxygenase (COX)-2 in LPS-induced RAW264.7 cells. The protein expression of nuclear NF-κB was measured by Western blot. The results showed that the safe medication range of SMP was less than 3 mg/mL. Compared with the LPS model group, SMP (2, 1, and 0.5 mg/mL) improved the degree of cell deformation and reduced the amount of pseudopodia, and statistically reduced the secretions of cytokines in cells induced by LPS (P < 0.01) at different time points. SMP significantly inhibited the mRNA transcriptions of TNF-α, IL-6, iNOS, and COX-2 and the protein expressions of NF-κB, p-p65, and p-IκBa. In conclusion, this study preliminarily proved the protective effect of SMP on LPS-induced RAW264.7 macrophage. Its mechanism might be related to inhibition of NF-κB signal pathway and the gene expressions and secretion of cytokines.
本研究探讨了丹参多糖(SMP)在脂多糖(LPS)刺激的 RAW264.7 细胞中的抗炎作用及可能的作用机制。采用 MTT 法检测 SMP 的细胞毒性。采用 Diff-Quik 染色观察 RAW264.7 细胞的形态变化。采用酶联免疫吸附试验(ELISA)检测 LPS 诱导的 RAW264.7 细胞中细胞因子的产生。采用一氧化氮(NO)试剂盒检测 LPS 诱导的 RAW264.7 细胞中 NO 的释放。实时聚合酶链反应(PCR)检测 LPS 诱导的 RAW264.7 细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、诱导型一氧化氮合酶(iNOS)和环氧化酶(COX)-2 的转录。采用 Western blot 检测核 NF-κB 的蛋白表达。结果表明,SMP 的安全用药范围小于 3mg/ml。与 LPS 模型组相比,SMP(2、1 和 0.5mg/ml)在不同时间点改善了细胞变形程度,减少了伪足数量,并且统计学上降低了 LPS 诱导的细胞因子分泌(P<0.01)。SMP 显著抑制 TNF-α、IL-6、iNOS 和 COX-2 的 mRNA 转录以及 NF-κB、p-p65 和 p-IκBa 的蛋白表达。综上所述,本研究初步证明了 SMP 对 LPS 诱导的 RAW264.7 巨噬细胞的保护作用。其机制可能与抑制 NF-κB 信号通路以及细胞因子的基因表达和分泌有关。