黄芪总黄酮对巨噬细胞RAW264.7抗炎免疫的双向调节研究

Study on the bi-directional regulation of anti-inflammatory immune response by total flavonoids of Astragalus in macrophages RAW264.7

为研究黄芪总黄酮(TFA)对巨噬细胞RAW264.7的抗炎免疫双向调节作用,本研究首先采用MTT法筛选TFA对RAW264.7细胞的安全剂量;后续研究均采用以下两种方式开展:采用不同安全剂量(10μg/mL、25μg/mL、100μg/mL)的TFA与正常培养的RAW264.7细胞作用;上述不同浓度的TFA与RAW264.7细胞作用后以终浓度为1μg/mL LPS刺激。分别通过ELISA和RT-PCR检测上述两种情况下RAW264.7细胞中细胞因子的含量及其mRNA的转录水平;采用western blot测定上述两种情况下RAW264.7细胞NF-κB信号通路中关键蛋白的表达水平。结果显示,TFA在0~150μg/mL对细胞无明显毒性;ELISA和RT-PCR结果显示,TFA能够显著提高正常培养的RAW264.7细胞上清液中IL-1β、IL-6和TNF-α含量及其mRNA转录水平,抑制LPS刺激的RAW264.7细胞上清液中IL-1β、IL-6和TNF-α的过度表达及其mRNA水平的过度转录,并呈剂量依赖性;western blot结果表明,TFA能够显著下调正常培养的RAW264.7细胞中IKKα/β、IκBα和C-NF-κB p65蛋白表达而上调p-IKKα/β、p-IκBα和N-NF-κB p65蛋白的表达,促进LPS刺激的RAW264.7细胞中IKKα/β、IκBα和C-NF-κB p65蛋白表达而抑制p-IKKα/β、p-IκBα及N-NF-κB p65蛋白的过度表达,并呈剂量依赖性。上述结果表明,TFA可分别通过调节正常和LPS刺激的RAW264.7细胞中关键蛋白的表达,适度活化NF-κB信号通路和抑制NF-κB信号通路的过度激活,提高正常培养的RAW264.7细胞细胞因子含量及mRNA转录水平,抑制LPS刺激的RAW264.7细胞促炎因子含量及mRNA水平的过度转录,从而发挥其抗炎免疫双向调节作用。本研究为TFA的临床应用提供参考依据和抗炎免疫药物的研发奠定基础。

In order to study the anti-inflammatory and immunomodulatory effects of total flavonoids of Astragalus(TFA) on macrophages RAW264.7, MTT method was used to screen the safe dose of TFA on RAW264.7 cells;the following two methods were used: RAW264.7 cells were treated with different doses of TFA(10 μg/mL, 25 μg/mL, 100 μg/mL), and cells were then stimulated with 1 μg/mL LPS. The levels of cytokines protein and mRNA in RAW264.7 cells were detected by ELISA and RT-PCR, and the expression levels of several key proteins in NF-κB signaling pathway were determined by western blot. The results showed that TFA had no obvious toxicity on RAW264.7 cells at the concentration of 0~150 μg/m L. ELISA and PT-PCR results showed that TFA significantly increased the expression levels of IL-1β, IL-6 and TNF-α proteins and m RNA transcripts in RAW264.7 cells, and inhibited the upregulated expression of IL-1β, IL-6 and TNF-α in RAW264.7 cells stimulated by LPS in a dose-dependent manner.Western blot results showed that TFA significantly down-regulated the expression of IKKα/β, IκBα and C-NF-κB p65 proteins and up-regulated the expression of p-IKKα/β, p-IκBα and N-NF-κB p65 proteins in RAW264.7 cells;while TFA promoted the expression of IKKα/β, IκBα and C-NF-κB p65 proteins and inhibited the upregulated expression of p-IKKα/β, p-IκBα and N-NF-κB p65 proteins in LPS-stimulated RAW264.7 cells. These results indicate that TFA can moderately activate the NF-κB signaling pathway and inhibit the its over-activation by regulating the expression of key proteins in normal and LPS-stimulated RAW264.7 cells, and increase the production and mRNA transcription of cytokines in normal cultured RAW264.7 cells, and inhibit the excessive production and mRNA transcription of pro-inflammatory cytokines in LPS-stimulated RAW264.7 cells, which exerts its bidirectional regulatory effects of anti-inflammatory immune response. This study providesa reference for the clinical application of TFA and lays a foundation for the research and development of anti-inflammatory drugs.