基于“既病防变”理论紫草素调控Hippo-YAP信号通路抗银屑病的作用机制研究

Psoriasis is one of the common chronic inflammatory dermatosis which affects people widely, but its treatment is not ideal. Traditional Chinese medicine Radix Arnebiae seu Lithospermi demonstrates remarkable therapeutic effect towards psoriasis. However，little information is available regarding the anti-psoriasis mechanism of its active component shikonin. Under the guidance of " preventing disease from exacerbating " of traditional theory and psoriasis pathogenesis of “hot blood”, we hypothesize that shikonin in anti-psoriasis may start on the early course of immune activation based on our team’s preliminary finding, which displayed shikonin significantly increasing psoriasis YAP expression level. Thus, Hippo - YAP signaling pathways may play an important role on early immune cell activation for psoriasis. In this project, we design in vivo and in vitro to detect expression level of YAP/RASSF, CD8+T cell activation and inflammatory cytokine intervened by a variety of concentration of shikonin. Furthermore，the regulation of the signaling pathway of Hippo-YAP by shikonin will be explored. Finally，the mechanism of shikonin affecting the early stage of activating RAS and CD8+T cell through the Hippo-YAP signaling pathway will be analyzed. Objectively, the study finding will not only provide theoretical basis for prevention and treatment of psoriasis, but also develop empirical evidence for searching anti-psoriasis drug targets.

银屑病为慢性炎症性皮肤病，累及人群广，常规治疗不理想。中医药治疗银屑病具有优势，处方中重用紫草可显著提高疗效。紫草素是紫草的主要成分，其抗银屑病作用机制不清。本课题组在“既病防变”理论和银屑病“血热”病机指导下提出，紫草素的抗银屑病作用可能启动于免疫活化的早期阶段。已证实紫草素可显著提高银屑病皮损中YAP的表达水平，而Hippo-YAP信号通路对早期免疫活化起重要作用。本项目拟从动物模型及细胞水平考察紫草素对YAP/RASSF的表达、CD8+T细胞的活化以及早期炎性因子/趋化因子的调控作用；继而体外RNA干扰HEK-α细胞中YAP/RASSF，深入研究紫草素在Hippo-YAP信号通路中的具体作用路径；分析紫草素通过Hippo-YAP信号通路干预RAS和CD8+T细胞的活化产生更早期的抗银屑病作用的机制，为银屑病的预防和临床干预提供理论基础，也为发现抗银屑病的药物靶点提供理论及实验依据。

银屑病是一种常见的免疫介导的慢性炎症性皮肤病，累及人群广泛。本病常规治疗效果不理想，极易复发。应用中医药治疗银屑病，疗效确切，不易复发。紫草以紫草素为主要成分。因此，本课题即研究紫草素的抗银屑病作用机制。本项目利用小鼠模型以及HaCaT细胞株模型，观察紫草素是否对小鼠模型的皮损、组织病理、小鼠模型和细胞模型中的炎性因子TNF-α、IL-17、IL-22和趋化因子CXCL1、CXCL2、S100A7表达水平以及RASSF、YAP、TAZ等Hippo-YAP信号通路相关因子表达水平有影响。实验结果显示，紫草素能明显改善小鼠模型的皮损外观及组织病理改变；能明显降低小鼠模型皮损中TNF-α、IL-17、IL-22和CXCL1、CXCL2、S100A7的表达水平；对小鼠模型皮损组织中的Hippo-YAP/TAZ信号通路的下游因子YAP和TAZ以及上游因子RASSF1A均有调控作用，能够上调RASSF1A基因RNA的表达，下调YAP和TAZ基因RNA的表达；对M5因子诱导的HaCaT细胞的炎性因子IL-17、IL-22、TNF-α和炎性趋化因子CXCL1、CXCL2、S100A7的表达有抑制作用；对Hippo-YAP信号通路中Mst1、Lats1/2、YAP、RASSF1A的磷酸化均有促进作用；对YAP沉默的M5因子诱导的HaCaT细胞的炎性因子IL-17、IL-22、TNF-α和炎性趋化因子CXCL1、CXCL2、S100A7的抑制作用均弱化，表明紫草素对Hippo-YAP信号通路中下游因子YAP有靶向调控作用。本研究在“既病防变”中医理论指导下，研究了紫草素通过调控Hippo-YAP信号通路阻断银屑病早期病程的具体作用机制，具有中医理论特色和创新性，为银屑病的临床预防和干预提供理论基础，对中医从血论治治疗银屑病的中医理论提供了科学的阐释。