黄芪多糖对THP-1源性巨噬细胞TLR4、Syk、Erk及Paxillin蛋白磷酸化水平与细胞吞噬功能的影响

Effects of astragalus polysaccharide on phosphorylated level of TLR4, Syk, Erk, Paxillin and phagocytic function of THP-1-derived macrophage

目的:观察黄芪多糖对"轻度氧化修饰低密度脂蛋白(mm LDL)-Toll样受体4(TLR4)-巨噬细胞"途径的干预作用,探讨其稳定动脉粥样硬化(AS)斑块、抑制AS进展的分子机制。方法:将THP-1源性巨噬细胞分为空白组,模型组,黄芪多糖低、中、高剂量。采用免疫印迹法检测脾酪氨酸激酶(Syk)、细胞外信号调节激酶(Erk)、桩蛋白(Paxillin)磷酸化水平随时间变化情况,以及不同剂量黄芪多糖对Syk、Erk、Paxillin蛋白磷酸化水平的影响;采用免疫沉淀联合免疫印迹法检测TLR4蛋白磷酸化水平随时间变化情况,以及不同剂量黄芪多糖对TLR4蛋白磷酸化水平的影响;采用中性红吞噬实验检测黄芪多糖对巨噬细胞吞噬功能的影响。结果:TLR4、Syk、Erk、Paxillin分别于15、10、15、30min达到磷酸化水平高峰;与空白组比较,模型组TLR4、Syk、Erk、Paxillin磷酸化水平升高(P<0.05)、细胞吞噬功能增强(P<0.05);与模型组比较,黄芪多糖低、中、高剂量组TLR4、Syk、ERK、Paxillin磷酸化水平降低(P<0.05)、细胞吞噬功能减弱(P<0.05);其中,黄芪多糖高剂量组上述改变明显强于黄芪多糖低、中剂量组(P<0.05)。结论:黄芪多糖可能通过"mm LDL-TLR4-巨噬细胞"途径影响巨噬细胞吞噬功能从而具有稳定AS斑块、干预AS作用。

Objective： To observe the effects of astragalus polysaccharide（APS） on the pathway of mild oxidative modification of low density lipoprotein-Toll like receptor 4-macrophage, and to explore its possible anti-atherosclerosis mechanism. Methods： THP-1-derived macrophage cells were divided into control group, model group, APS low-dose group, APS middle-dose group, and APS high-dose group. Western blot was used to detect the proteins expression of p-Syk, p-Erk, p-Paxillin in different time and groups. Western blot combined immunoprecipitation were used to detect the protein expression of p-TLR4 in different time and groups. Neutral red phagocytosis test was used to measure the effect of APS on phagocytic function of macrophage cells. Results： TLR4, Syk, Erk and Paxillin reached the peak of phosphorylation at 15, 10, 15, 30 min after intervention, respectively. Compared with the control group, the proteins expression of p-TLR4, p-Syk, p-Erk and p-Paxillin in the model group and APS intervention groups were increased（P〈0.05）, as well as phagocytic function of macrophage cells（P〈0.05）. Compared with model group, the proteins expression of p-TLR4, p-Syk, p-Erk and p-Paxillin in all of APS intervention groups were decreased（P〈0.05）, as well as phagocytic function of macrophage cells（P〈0.05）. There were statistical differences in changes in these observation targets between the APS high-dose group and both of the APS low-dose group and APS middle-dose group（P〈0.05）, with the APS high-dose group changing significantly（P〈0.05）. Conclusion： Astragalus polysaccharide can inhibit phagocytic function and have the anti-atherosclerotic effect through the pathway of mild oxidative modification of low density lipoprotein-Toll like receptor 4-macrophage.