党参多糖对巨噬细胞的诱导活化作用

doi:10.11843/j.issn.0366-6964.2017.04.022

摘要/Abstract

摘要：

旨在通过检测党参多糖（Codonopsis pilosula polysaccharide，CPPS）对小鼠巨噬细胞系RAW 264.7细胞的增殖作用、TNF-α和IL-6分泌及其mRNA转录和NF-кB通路活化情况，探究CPPS对小鼠巨噬细胞系的诱导活化作用。以水提醇沉法从山西道地药材潞党参提取CPPS，经Sevage法除蛋白质，苯酚-硫酸法测定其含量。采用MTT法检测不同质量浓度CPPS（从31.25到4 000 μg·mL^-1以2倍倍比稀释的方式）对RAW 264.7细胞增殖的影响。分别将25、50、100、150和200 μg·mL^-1的CPPS作用于巨噬细胞系RAW 264.7细胞，ELISA检测TNF-α和IL-6的分泌量，RT-PCR检测TNF-α和IL-6 mRNA转录量，Western blot检测细胞核内NF-кB p65和细胞质内IкB-α蛋白表达量。结果显示，CPPS含量为83.97%；CPPS质量浓度在31.25～250 μg·mL^-1可刺激RAW 264.7细胞增殖，以125 μg·mL^-1增殖作用最显著（P<0.01），而高于500 μg·mL^-1时则抑制RAW 264.7细胞的增殖；100和150 μg·mL^-1的CPPS可使TNF-α（P<0.01）和IL-6（P<0.05）的分泌量及其mRNA的转录量极显著增加（P<0.001）；100 μg·mL^-1的CPPS可刺激RAW 264.7细胞核内NF-кB p65蛋白表达量升高（P<0.01），并使细胞质内IкB-α蛋白表达量降低（P<0.001）。结果表明，CPPS不仅可以通过刺激RAW 264.7细胞增殖，还可能通过激活NF-кB信号通路使TNF-α和IL-6的分泌量增多，进而参与机体的免疫调节。

Abstract:

The RAW264.7 cells activation of Codonopsis pilosula polysaccharide (CPPS) was investigated through the detection of cell proliferation, TNF-α and IL-6 secretion and mRNA transcription, NF-кB signaling pathway activation. CPPS were obtained by water extraction and ethanol precipitation, then proteins were removed by Sevage method and the content was determined by Phenol-Sulfuric acid method. The proliferation of RAW264.7 cells incubated with different concentrations CPPS (2-fold serially diluted from 31.25 to 4 000 μg·mL^-1) was detected by MTT method. RAW264.7 cells incubated with various concentrations of CPPS (25, 50, 100, 150 and 200 μg·mL^-1), TNF-α and IL-6 secretion were measured using ELISA kits; The TNF-α and IL-6 mRNA transcription were evaluated by RT-PCR. As well, expression of NF-кB p65 and IкB-α was determined by Western blot. The content of CPPS was 83.97%; CPPS (31.25-250 μg·mL^-1) exerted proliferation effect on RAW264.7 cells, and the CPPS concentration of 125 μg·mL^-1 proliferated significantly (P<0.01). While the concentration of CPPS higher than 500 μg·mL^-1 inhibited the proliferation of RAW 264.7 cells. The CPPS (100 and 150 μg·mL^-1) could increase the secretion of TNF-α (P<0.01) and IL-6 (P<0.05), and significantly increased the transcription of TNF-α (P<0.001) and IL-6 (P<0.001). Moreover, CPPS concentration of 100 μg·mL^-1increased NF-кB p65 expression in nuclear (P<0.01) and decreased IкB-α expression in cytoplasm (P<0.001). CPPS can not only stimulate the RAW264.7 cells proliferation, possibly through activation of NF-кB signaling pathway to the secretion of TNF-α and IL-6 increased, which are involved in immune regulation.

中图分类号:

石轶男, 孙娜, 孙耀贵, 李宏全. 党参多糖对巨噬细胞的诱导活化作用[J]. 畜牧兽医学报, 2017, 48(4): 777-784.

SHI Yi-nan, SUN Na, SUN Yao-gui, LI Hong-quan. Codonopsis pilosula Polysaccharide-induced Activation and Proliferation of Macrophage RAW264.7 cell[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(4): 777-784.

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