基于激活PPARγ—阻断DC/Th17/Th22探讨土槿乙酸对特应性皮炎的免疫干预作用及机制研究

Atopic dermatitis (AD) is a kind of chronic allergic dermatitis mediated by T lymphocytes, which decreases the patients' life quality seriously. At present, the efficiency of common drugs for AD are limited and certain side effects remain as a major obstacle. It's well known that dendritic cell(DC) and T helper cell subsets (Th17, Th22 ) could paly important roles in the pathogenic process of AD. Recently, it's reported that peroxisome proliferator-activated receptor γ(PPARγ) could down-regulate DC/Th17/Th22 cells to inhibit the pathogenesis of AD. The root and trunk bark(pseudolarix) of Pseudolarix amabilis (Nelson) Rehd. endemic to China is a type of conventional chinese medicine for dermatosis. Pseudolaric acid B (PB), an active ingredient of Pseudolarix, is a natural agonist for transcription activation of PPARγ, which activity is superior to some other synthetic agonists. We have firstly proved that PB had relatively higher immunosuppressive activity both in vitro and in vivo, with the inhibitory effect to the proliferation of antigen-specific T cells. Moreover, PB could inhibit the production of Th17. Further studies have indicated that PB could relieve the pathogenic process of AD significantly in mouse model with high safety. However, the mechanism of PB is needed to be further evaluated. Now, there have not been related research on the effects of PB to AD at home and abroad. Thus, we hypothesize that PB might interfere the pathogenesis of AD via activating PPARγ and suppressing DC/Th17/Th22 cells. In order to confirm the hypothesis, we would investigate the effects and mechanism of PB to related cell subsets and signal transduction pathway in animal model and cells evaluation system, which could be carried out from the level of entire animal,cell and molecule. In our subject, we hope to lay a foundation for finding some new targets of PB and developing some new chinese medicine to treat and prevent AD with high efficiency and low toxicity.

特应性皮炎（AD）是T细胞介导的慢性过敏性皮肤病，严重影响患者生活质量。常用治疗药物均不理想且毒副作用较多。过氧化物酶体增殖物活化受体γ（PPARγ）可通过负调节参与AD发病的重要免疫细胞DC/Th17/Th22抑制AD。皮肤科传统中药土槿皮为中国特有植物金钱松根皮，其有效成分土槿乙酸（PB）是高活性的天然PPARγ激动剂。我们前期首次发现，PB体内外均具良好的免疫抑制活性，还能抑制抗原特异性T细胞活化增殖，下调Th17产生，明显缓解小鼠AD表现，且安全性高，但具体机制有待进一步深入研究。国内外尚未见PB对AD作用的报道。我们推测"靶向激活PPARγ-阻断DC/Th17/Th22"可能是PB干预AD的重要途径。为证实假说，本项目拟通过动物模型和细胞评价体系，从整体-细胞-分子层次研究PB对相关细胞亚群和信号转导通路的影响，为发现PB作用的新靶点、研制高效低毒应用于AD的中药新药奠定基础。

特应性皮炎（AD）是是以皮肤高反应性为特征、由T细胞介导的常见慢性过敏性皮肤病，T 细胞介导的慢性过敏性皮肤病，严重影响患者生活质量。迄今为止，AD的确切病因和发病机理尚未完全阐明，病程易迁延慢性化，仍无理想治疗药物。皮肤科传统中药土槿皮为中国特有植物金钱松根皮，其有效成分土槿乙酸（PB）具有良好的抗炎免疫调节活性。本项目在前期工作的基础上，通过体外细胞培养和小鼠AD模型，探讨了PB免疫调节活性的具体机制，从其对不同T细胞亚群分化的影响，特别是从对Th22和Th17细胞影响的崭新视角探讨其效应机制，找到防治AD的新靶点，为免疫干预AD提供新的研究思路。. 本项目首先根据文献报道和预实验，摸索出建立小鼠AD模型的最佳方案，并应用淋巴细胞转化实验、组织形态观察、ELISA及PCR等方法对模型加以验证。应用MTT法、MLR检测PB的细胞毒性及其对正常小鼠T淋巴细胞和特异性T淋巴细胞体外增殖能力的影响，应用ELISA检测特异性T细胞免疫应答水平。分别经皮和灌胃给药干预，体内分析PB对AD小鼠的药效学作用，然后，进一步探讨PBB对AD小鼠Th17、Th22细胞分化及其相应细胞因子、核因子表达的影响，并根据基因芯片筛查结果着重从p38MAPK、NF-κB、PPARγ通路的角度研究PB的免疫调节作用机制，明确PB免疫干预AD的药物靶点。体内研究结果显示， PB能够剂量依赖的抑制AD小鼠耳肿胀，表皮及真皮水肿明显减轻，炎症细胞浸润显著减少；明显促进PPARγ的基因和蛋白表达；剂量依赖的促进PPARγ转录激活，且该作用能够被PPARγ特异性抑制剂GW9662所阻断；同时明显下调p38MAPK、MK-2、IKBα等信号分子的活性表达，抑制炎症小体NLRP3表达，提示其抗AD作用与调控MAPK-NK-κB-PPARγ通路密切相关。体外实验同样证实，PB可通过调控MAPK-NK-κB-PPARγ通路发挥免疫抑制作用，且细胞毒性低，具有优越的安全性。. 本项目从整体—细胞—分子层次研究 PB 对相关细胞亚群和信号转导通路的影响，为寻找新型抗AD药物，研制高效低毒应用于 AD 的中药新药奠定基础，丰富中西医结合治疗AD的内涵。