从调节HSP70-树突状细胞TLR4-NF-κB活化通路研究养血解毒汤治疗银屑病的分子机制

Dendritic cells（DC）Abnormal activation is the starting point of psoriasis onset, while HSP70 mediates activation of DC, and produces inflammatory immune response through HSP70/TLR4-NF-κB signaling pathway,release Th1 type inflammatory factors. triggering, continuing and aggravating the disease.The course of psoriasis of blood dryness syndrome type is long and unhealed, which is the difficult point of clinical treatment. The pathogenesis of psoriasis is mainly dryness and dampness alternation, dampness toxin endogenetic, causing abnormal dressing in body fluid, which is closely related to the pathophysiological changes of psoriasis.The method of nourishing blood and detoxifying was used in clinic, and the prescription of nourishing blood and detoxification showed good clinical effect.The prediction of effective components of Yangxue jiedu recipe by network pharmacological target had certain correlation with HSP70.Studies have shown that HSP70 can induce DC activation and produce Th1 type inflammatory response through the TLR4-NF-κB signaling pathway.According to the study, HSP70 can not only induce DC activation, but also induce the immune response of cells significantly, and can be used as a target protein to treat psoriasis. Therefore, this study attempts to use HSP70 as the central link of pathogenetic mechanism of psoriasis. To break the loop of "dryness and dampness interconversion, dampness toxin endogenetic", deeply explain the pathogenesis characteristics and therapeutic targets of psoriasis blood-dryness syndrome;The effects of Yangxue jiedu recipe on DC differentiation, maturation, function and TLR4 signal transduction pathway related proteins were observed in vitro. The pathogenesis of psoriasis blood-dryness syndrome was elucidated from animal, cell and molecular levels, which provided scientific basis for clinical treatment.

树突状细胞（DC）的异常活化是银屑病发病的起始环节，皮肤应激条件下产生的热休克蛋白70家族以外泌体形式通过TLR4-NF-κB通路活化树突状细胞，释放Th1型炎症因子，触发并维持皮损炎症反应。银屑病血燥证病程绵长，其病机为燥湿互化，湿毒内生，致津液敷布异常，肌肤失养。临床上养血解毒方显示良好的疗效且前期网络药理靶点预测养血解毒方有效组分与HSP70有一定的关联性。因此本研究尝试以HSP70作为银屑病致病病理机制的中心环节，打破“燥湿互化，湿毒内生”的环路，诠释银屑病血燥证病机特点及治疗靶点；并体内外观察养血解毒方对银屑病样动物及DC活化和HSP70介导的DC-TLR4-NF-κB信号通路相关蛋白的影响，从动物、细胞、分子水平阐明银屑病血燥证病机基础，为临床治疗提供科学依据。

本课题拟观察养血解毒汤对咪喹莫特诱导小鼠银屑病样皮损模型和细胞培养两个层面，深入探究了养血解毒法作用机制是否通过抑制HSP70 阻断树突状细胞 HSP70/TLR4-NF-κB 活化通路。通过动物和细胞学研究的结果相互验证，阐明养血解毒汤对血燥型银屑病的疗效机制，从而阐明血燥证燥湿互化，湿毒内生的病机实质，提升养血解毒法的科学内涵，为临床用药提供理论指导。通过研究发现：①咪喹莫特乳膏外涂于BALB/c小鼠背部皮肤5天可诱导出银屑病样小鼠模型，皮疹表现及病理改变与银屑病相似。②养血解毒汤、甲氨蝶呤和HSP70抑制剂小鼠皮损改变较模型组不同程度好转，其中甲氨蝶呤组效果最佳。③模型组小鼠皮肤中PCNA、Ki67、HSP70、TNF-α, IL-6, IL-1β, IFN-γ, IL-17 and IL-23及蛋白表达水平均较空白组明显升高，各治疗组均较模型组呈不同程度降低。④养血解毒汤、甲氨蝶呤和HSP70抑制剂各组均能改善免疫T细胞的表达，抑制TLR4-NF-κB信号通路的活化。⑤通过IFN-γ、LPS刺激HaCaT细胞建立角质形成细胞活化和炎症的银屑病体外细胞学模型，最佳刺激因素可选择IFN-γ50ng/ml、LPS 2ug/ml刺激12小时。⑥含药血清和甲氨蝶呤对银屑病模型中细胞因子HSP70、TNF-α，IL-6，IL-17，IL-23有一定抑制作用，且可抑制TLR4，NF-κB p65的表达，阻碍加IκBα的降解，与HSP70抑制剂组效果趋势一致，说明养血解毒汤可能通过抑制HSP70基因表达，抑制TLR4-NF-κB信号通路的活化，进而降低炎症因子的分泌，从而发挥治疗银屑病的作用。综上所述，HSP70、TLR4/NF-κB信号通路可能参与银屑病的发病过程。其中HSP70可能成为银屑病治疗的有效靶点。养血解毒汤是治疗血热型寻常性银屑病的有效方剂，该研究内容为今后养血解毒汤应用于临床提供科学依据。针对血燥型病因病机，治以祛湿解毒，养血润肤，并通过调控HSP70的水平从而抑制TLR4/NF-κB通路活性，继而抑制炎症因子的表达，进而发挥治疗银屑病的作用。