羟基红花黄色素A抑制LPS激活TLR4受体的机制研究

It is shown that Toll like receptor 4 (TLR4) is related to pathogenesis in many inflammatory diseases. Lipopolysaccharide (LPS) is the specific ligand of TLR4. We have found that Hydroxysafflow yellow A (HSYA) can inhibit NF-κB activation and inflammatory factor expression induced by LPS. However the target protein of HSYA in the LPS-TLR4-NFκB signaling pathway is still uncertain. It is important to study the mechanism of HSYA on inhibiting LPS activating TLR4, which will provide crucial proof to cure TLR4 related inflammatory diseases. According to our research results and pharmacology character of HSYA, we want to find the target protein of HSYA from LBP, CD14, MD-2 and TLR4 which involve in LPS activating TLR4. We will employ experiments to compare the binding character of HSYA with proteins, effect of HSYA on protein structure and inhibition on proteins binding to LPS. To verify the target protein, we research the effect of HSYA on initial stage of TLR4 activation such as TLR4 dimerization and recruiting adaptor proteins. We examine the inhibition of HSYA on LPS activating TLR4 by observing NF-κB and IRF3 nuclear factors activation in cell and mice inflammatory response models induced by LPS.

Toll样受体4（TLR4）参与多种炎症性疾病的病理过程，脂多糖（LPS）是其特异性配体。我们发现羟基红花黄色素A（HSYA）有抑制LPS诱导的NF-κB激活和炎症因子表达的作用，但HSYA在LPS-TLR4-NFκB信号通路上作用的靶蛋白尚不明确，阐明HSYA抑制LPS激活TLR4的机制，将会对与TLR4相关的炎症性疾病的治疗提供理论依据。根据前期实验和HSYA作用特点，拟从LPS激活TLR4过程的四种相关蛋白LBP、CD14、MD-2和TLR4分子中，通过比较HSYA与四种蛋白的结合动力学、HSYA对四种蛋白构象的影响、抑制四种蛋白与LPS结合功能等发现HSYA的作用靶蛋白。并以HSYA抑制TLR4二聚化和抑制募集接头蛋白等TLR4激活起始环节来验证靶蛋白。在LPS诱导的细胞和小鼠炎症模型中，通过观察对核转录因子NF-κB和IRF3激活，检验HSYA抑制LPS激活TLR4的作用。

TLR4诱导的炎症反应增加，是许多炎症性疾病发生发展的重要机制之一。TLR4与细菌外膜成分LPS信号转导的活化密切相关。细胞表面的TLR4接受LPS的刺激，激活转录因子NF-κB，从而释放一系列炎症因子。本项目主要研究了羟基红花黄色素A(HSYA)抑制LPS激活TLR4的炎症反应，发现了HSYA可从多个途径减弱TLR4受体信号通路激活的部分机制。在体外模型，发现HSYA可能与CD14和MD-2蛋白分子形成复合物，干扰FITC-LPS与细胞的结合。在培养细胞水平，抑制LPS诱发的人外周血白细胞与培养内皮细胞的体外粘附；HSYA可以缓解LPS所致培养细胞上清液中粘附分子ICAM-1及炎性因子TNF-α、IL-1β 和 IL-6的表达升高；HSYA可以降低LPS诱发的TLR-4及MyD88表达的升高与NF-κBp65的核转位以及p38MAPK蛋白磷酸化水平的升高；HSYA可以抑制LPS所致TLR-4、MyD88、ICAM-1及TNF-α、IL-1β 和 IL-6 mRNA 表达的升高； HSYA可以抑制LPS所致NF-κB转录因子的活化。在动物模型，HSYA可以缓解小鼠肺部炎症损伤引起的体重减轻、左肺系数增加、炎症细胞浸润和肺组织结构的破坏；HSYA可以缓解炎症小鼠早期肺部少量的胶原沉积。HSYA能抑制LPS诱导的小鼠肺组织p65和α-SMA表达水平升高。小鼠血浆中TGF-β1，IL-1β蛋白表达升高也明显受到抑制。HSYA能抑制LPS所致炎症小鼠肺组织中TLR4, CD14, MD-2, Col I 和α-SMA蛋白表达升高。LPS模型小鼠炎性因子（TLR4, CD14, TNF-α, IL-1β, IL-6）和致纤维化相关因子（TGF-β1, α-SMA, Collagen I, Collagen III）mRNA的表达水平明显升高，给予不同剂量的HSYA后上述细胞因子的mRNA 水平升高明显受到抑制。综上所述，HSYA缓解小鼠炎症的作用机理可能是通过干扰LPS与细胞的结合，以及抑制信号相关蛋白的表达，从而抑制TLR4/NF-κB介导的炎症信号转导。HSYA可从不同环节来抑制炎症因子活化的级联反应，其抑制炎症反应机理的进一步阐明，有助于该药物在临床相关领域的开发应用。