苦参甘草组方对肝脏星状细胞和库普弗细胞炎症通路的双重抑制作用及其分子机理研究

Chronic liver injury is mainly induced by activating the signal pathway of TGF-βin hepatic stellate cell (HSC) and TLR in Kuffer cell. Our previous studies suggested that the flavescen and liquorice prescription (FLP) exhibited stronger hepatic protection and weaker adverse reactions than the administration of flavescens (Fla) or liquorice (Liq) alone. This might be related to the inhibition of inflammation response mediated by HSC and Kuffer cell. In this study, C57BL/6 mice,Smad konckout mice and MyD88 knockout mice are selected to establish the model for CCl4-induced chronic liver injury. The differences on hepatic protection between FLP and Fla or Liq will be analyzed. On the same time, we will also explore the differences of Fla effects between C57BL/6 mice and Smad knockout mice as well as the differences of Liq effects between C57BL/6 mice and MyD88 knockout mice. We hope to illuminate the biphasical inhibition of flavescen and liquorice prescription on hepatic inflammation pathways mediated by Kupffer and HSC and its molecular mechanism. This work would provide a new idea for the investigations of traditional Chinese medicine, which not only opposes each other but also complements each other. At the same time, our studies theoretically offer the basis for the modernization of flavescen and liquorice prescription and the treatment of chronic hepatic diseases.

慢性肝损伤主要由肝脏星状细胞TGF-β信号传导途径和库普弗细胞TLR信号传导途径激活导致。我们发现，苦参甘草组方与单独使用苦参或甘草相比，肝脏保护作用增强，不良反应下降。该作用与抑制星状细胞和库普弗细胞介导的炎症反应有关。本项目拟采用C57BL/6小鼠、Smad基因敲除小鼠及MyD88基因敲除小鼠，建立CCL4慢性肝损伤模型，比较苦参甘草组方与单独使用苦参或甘草时，对C57BL/6小鼠肝脏保护作用的差异；单独使用苦参时， 比较C57BL/6小鼠与Smad基因敲除小鼠肝脏保护作用的差异；单独使用甘草时，比较C57BL/6小鼠与MyD88基因敲除小鼠肝脏保护作用的差异。本研究旨在：阐明苦参甘草组方对肝脏星状细胞和库普弗细胞炎症通路的双重抑制作用及其分子机理；为中药组方"相须为用"作用特点的研究，提供了一种新的思路；为苦参甘草组方的中药现代化及其在慢性肝脏疾病治疗中的应用提供理论依据。

慢性肝损伤主要由肝脏星状细胞TGF-β信号传导途径和库普弗细胞TLR 信号传导途径激活导致，我们既往研究发现，苦参甘草组方与单独使用苦参或甘草相比，肝脏保护作用增强，不良反应下降，该作用与抑制星状细胞和库普弗细胞介导的炎症反应及纤维化有关。本课题研究旨在通过动物及细胞模型实验：阐明苦参甘草组方对肝脏星状细胞和库普弗细胞信号传导途径的双重抑制作用及其分子机理。本研究通过采用急慢性肝损伤动物模型及细胞模型，考察了苦参甘草组方在动物及细胞模型中对肝脏及肝细胞的保护作用，同时通过实时定量PCR，免疫组化，免疫蛋白定量与HPLC-MS联用技术等考察了苦参甘草组方对动物及细胞模型TGF-β信号传导途径和TLR 信号传导途径的影响，明确了该组方保护肝损伤的分子机制，实验结果表明：苦参甘草组方对肝脏中TGF-β/Smad纤维化信号途径及TLR4/Myd88炎性途径具有双重抑制作用，与课题理论假设一致；苦参甘草组方中：①甘草中的甘草甜素通过参与调控TLR4/Myd88炎性途径，发挥抗炎作用；②苦参中的苦参碱通过TGF-β/Smad信号途径抑制肝纤维化的形成。此外研究发现甘草中甘草甜素可以通过抑制TGF-β/PI3K/Akt纤维化信号途径，发挥抑制抗肝纤维化发生的作用。该调控作用与TGF-β蛋白表达有关，但与Smad无关。本课题研究的完成，为中药组方“相须为用”作用特点的研究，提供新的思路；为苦参甘草组方在慢性肝脏疾病治疗中的应用提供理论依据。