基于TLR4信号通路的黄芪总黄酮抗炎免疫调节机制研究

Radix Astragali has invigorating vital energy and splenic yang, strengthening exterior and reducing sweat, pusdraining and toxin-expelling, inducing diuresis to alleviate edema, astringing sores and promoting granulation effects. It has been widely used in veterinary clinic. The total flavonoids of Astragalus (TFA) is one of the main active components of Radix Astragali, but the study on anti-inflammation and immunoregulation of TFA is less, its molecular mechanism is still blank. First, the effects of TFA on cellular immune function, nuclear factor AP-1 and NF-κB activities, related protein expressions or phosphorylation levels, and toll like receptor 4 (TLR4) gene expression in normal and LPS-stimulated macrophages will be studied in vitro via signal transduction pathway. Adding specific TLR4 pathway blocking agents, the effects of TFA on macrophage function and signal transduction pathway will be further verified, and determine the molecular mechanism and effective target of TFA. Based on this, the in vivo anti-inflammatory and immunoregulatory effects of TFA will be tested in normal and inflammatory animal models. It will provide theoretical direction and technological support for scientific application of TFA as a lead compound in veterinary clinic, also establish a new pathway for new drug target screening and molecular level study of traditional Chinese medicines.

黄芪具有补气升阳、固表止汗、托毒排脓、利水消肿、敛疮生肌之功效，在兽医临床上已广泛应用。黄芪总黄酮为黄芪的主要活性成分之一，关于其抗炎免疫调节作用的研究较少，分子机制的研究尚属空白。本课题首先借助信号转导通路平台通过体外试验研究黄芪总黄酮对正常和LPS刺激的巨噬细胞免疫功能、核转录因子AP-1和NF-κB活性、TLR4通路中关键性蛋白表达或磷酸化水平及TLR4等基因表达的影响，再通过加入特异性TLR4通路阻断剂进一步验证其对巨噬细胞功能及信号转导通路调控的影响，从而确定黄芪总黄酮的抗炎免疫调节分子机制和作用靶标；在此基础上，通过正常和炎症动物模型试验，验证黄芪总黄酮的体内抗炎和免疫调节作用。这将为黄芪总黄酮作为先导化合物在兽医临床上的科学应用提供理论指导和技术支撑，也为新的药物靶标筛选和中药的分子水平研究开辟一条新途径。

黄芪具有补气升阳、固表止汗、托毒排脓、利水消肿、敛疮生肌之功效，在兽医临床上已广泛应用。黄芪总黄酮为黄芪的主要活性成分之一，关于其抗炎免疫调节作用的研究较少，分子机制的研究尚属空白。本课题首先借助信号转导通路平台通过体外试验研究了黄芪总黄酮对正常和LPS刺激的巨噬细胞免疫功能、核转录因子AP-1和NF-κB活性、TLR4基因表达等的影响，从而确定了黄芪总黄酮的抗炎免疫调节分子机制和作用靶标；在此基础上，通过正常和炎症动物模型试验，验证了黄芪总黄酮的体内抗炎和免疫调节作用。研究表明：黄芪总黄酮通过调控NF-κB信号转导通路中关键因子IKKα/β及p-IKKα/β、IκBα及p-IκBα、C-NF-κB p65及N-NF-κB p65等蛋白表达，和调控MAPKs信号传导通路中ERK1/2、p38等的磷酸化水平，上调了正常巨噬细胞细胞因子和介质含量、细胞因子和介质mRNA表达及相关酶iNOS/COX-2 mRNA和蛋白表达水平，下调了LPS刺激的巨噬细胞细胞因子和介质含量、其mRNA表达及相关酶mRNA和蛋白表达水平，从而发挥其抗炎免疫双向调节作用。并研究表明：黄芪总黄酮能通过提高小鼠单核巨噬细胞吞噬功能、血清溶血素水平，抑制小鼠迟发型超敏反应，促进小鼠脾淋巴细胞增殖，从而显著增强小鼠的免疫功能；通过调控OPG/RANKL/NF-κB通路对大鼠佐剂性关节炎具有明显的保护作用。这为黄芪总黄酮作为先导化合物在兽医临床上的科学应用提供理论指导和技术支撑，也为中药的分子水平研究开辟一条新途径。