基于TLR4/MyD88/NF-κB信号通路研究黄连素对小鼠巨噬细胞极化的干预作用

Intervention Effects of Berberine on Mice Macrophage Polarization Based on TLR4/MyD88/NF-κB Signaling Pathway

目的:基于Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核因子κB(NF-κB)信号通路研究黄连素对小鼠巨噬细胞极化的影响。方法:以小鼠巨噬细胞RAW264.7为对象,以阿托伐他汀钙为阳性对照,经脂多糖(LPS)诱导以复制炎症细胞模型,采用酶联免疫吸附测定法检测低、中、高剂量黄连素(5、10、20μmol/L)作用24 h后细胞培养液中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、NF-κB含量,采用实时荧光定量聚合酶链反应法检测细胞中TLR4、MyD88 mRNA的表达水平,采用Western blotting法检测细胞中TLR4、MyD88、诱导型一氧化氮合酶(iNOS)、CD206蛋白的表达水平。结果:与空白对照组比较,LPS诱导组细胞培养液中TNF-α、IL-6、NF-κB含量,细胞中TLR4、MyD88 mRNA的相对表达量以及TLR4、MyD88、iNOS蛋白相对表达量均显著升高(P<0.05)。与LPS诱导组比较,阿托伐他汀钙组和黄连素中、高剂量组TNF-α、IL-6含量,TRL4、MyD88 mRNA及其蛋白的相对表达量以及各给药组NF-κB含量和i NOS蛋白的相对表达量均显著降低,且黄连素高剂量组NF-κB含量显著低于阿托伐他汀钙组(P<0.05);阿托伐他汀钙组和黄连素高剂量组CD206蛋白的相对表达量均显著升高,且黄连素高剂量组CD206蛋白的相对表达量显著高于阿托伐他汀钙组(P<0.05)。结论:不同剂量的黄连素均可不同程度地干预小鼠巨噬细胞极化,其机制可能与调控TLR4/MyD88/NF-κB信号通路有关。

OBJECTIVE:To study the effects of berberine on mice macrophage polarization based on TLR4-MyD88-NF-κB signaling pathway. METHODS: Using mice RAW264.7 macrophage as the object, atorvastatin calcium as positive control,inflammatory cell model was induced by lipopolysaccharide(LPS);ELISA method was used to detect the contents of TNF-α,IL-6 and NF-κB in cell culture medium after treated with low,medium and high doses of berberine(5,10,20 μmol/L)for 24 h. The real-time fluorescence quantitative PCR was conducted to determine the mRNA expression of TLR4 and MyD88 in cells. Western blotting assay was used to detect the protein expression of TLR4,MyD88,iNOS and CD206 in cells. RESULTS:Compared with blank control group,the contents of TNF-α,IL-6 and NF-κB in cell culture medium,mRNA expression of TLR4 and MyD88,protein expression of TLR4,MyD88 and iNOS in cells were increased significantly in LPS induction group(P<0.05). Compared with LPS induction group,the contents of TNF-α and IL-6,mRNA and protein expression of TLR4 and MyD88 in atorvastatin calcium group,berberine medium-dose and high-dose groups as well as the content of NF-κB and protein expression of iNOS in administration groups were decreased significantly,while the content of NF-κB in berberine high-dose group was significantly lower than atorvastatin calcium group(P<0.05).The protein expressions of CD206 in atorvastatin calcium group and berberine high-dose group were increased significantly,while the protein expression of CD206 in berberine high-dose group was significantly higher than atorvastatin calcium group(P<0.05). CONCLUSIONS:Different doses of berberine can intervene in mice macrophage polarization to different extents,the mechanism of which may be associated with the regulation of TLR4/MyD88/NF-κB signaling pathway.