黑骨藤苷治疗AD及对TH2细胞分化和DC成熟作用的分子机制研究

Atopic dermatitis (AD), a chronic relapsing inflammatory skin disease, is increasing in prevalence around the world. AD is a disease with TH2 and TH22 cell polarization , but TH1 and TH17 cells are also involved in the development of AD pathology. Dendritic cells (DCs) activated by thymic stromal lymphopoietin (TSLP) prime naive CD4+T cells to differentiate into T helper type 2 (Th2) cells that play an important role in the development of AD. Periploca forrestil which is effective for treating RA has been used in clinical, Periploca forrestil contains cardiac glycosides, alkyl steroid. Periplocoside is one of its active ingredients. In previous study， we found that total glycosides of Periploca forrestil improve symptoms of dermatitis induced by oxazolone application repeatedly, and reduce mRNA expression of GATA3 in the lesinal skin, its detail mechanisms in treating AD is still less known. In this project，we will explore the effects of total glycosides of Periploca forrestil on the invasion of T cells, dendritic cell and the expression of factors related to skin barrier in OVA-induced AD-like skin in mice，and on the cytokine and transcription factors related to TH1/TH2/TH17/TH22 cell defferentiation. In vitro we will focus on the effects of total glycosides of Periploca forrestil and Periplocoside on differentiation of TH2 cell cultured in TH2 cell polerization condition, and binding activities of GATA3 and Gfi1. Then we will evaluate the effects of total glycosides of Periploca forrestil and Periplocoside on defferentiation of TH2 cells primed by TSLP-activated dendritic cells through measuring the TH2 cell type cytokines, and their effects on the molecular mechanisms of JAK/STAT pathway and NF-κB route in TSLP-activated dendritic cells. The evaluation through multi-target is in line with traditional Chinese medicine treatment mechanism. Research based on AD pathogenesis combined with the latest development on the study of immunology mechanism in atopic dermatitis treatment will provide experimental evidences for the clinical treatment of AD.

AD被认为是TH2和TH22细胞免疫反应介导的慢性复发性皮肤病， 但Th1和TH17细胞也参与AD发展。TSLP激活 DCs诱导TH2细胞分化在AD病中起重要作用。黑骨藤为祛风湿民族药。前期研究发现黑骨藤苷有改善噁唑酮诱导大鼠的皮肤炎作用。本项目研究黑骨藤苷及其有效成分杠柳苷对胶带粘贴和反复OVA攻击小鼠表皮诱导的AD样皮肤炎T细胞、树突状细胞、肥大细胞侵润以及与皮肤终末分化相关因子表达，以及AD皮肤细胞中TH1/TH2/TH17/TH22细胞相关因子和转录因子的作用；体外实验研究黑骨藤苷和杠柳苷对TH2细胞分化及其转录因子GATA3和Gfi1活性的作用；研究黑骨藤苷和杠柳苷对TSLP刺激DCs诱导的TH2细胞分化以及TSLP刺激DCS的JAK/STAT5和NF-κB的作用及其分子机制。本研究评价指标与AD致病机理结合，用免疫学最新理论研究中药治疗AD作用的多种机理，可为治疗AD提供依据。

黑骨藤为民族药，已被广泛用作治疗风湿病，关节痛和创伤。但是，其药理活性尚未完全阐明。.本研究旨在探索Periploca forrestii Schltr saponin（PFS）对噁唑酮（Oxazolone）诱导的特应性皮炎（AD）和巨噬细胞极化的作用及其潜在机制。体内实验采用噁唑酮反复涂布BALB/c小鼠的耳部皮肤诱导AD，PFS灌胃。取血浆和耳组织进行生物化学或组织学检测。体外模型采用脂多糖（LPS）刺激的小鼠骨髓来源的巨噬细胞（BMDM）极化。检测BMDM的增殖和蛋白质表达。PFS治疗抑制AD发展和血浆IgE的过量产生。 PFS改善耳组织中表皮厚度，炎症细胞浸润和一氧化氮合酶（NOS）2和精氨酸酶（Arg）1的表达。PFS治疗下调Th1（M1）因子MCP-1，TNF-α和IL-6，Th2（M2）因子如IL-4，Arg1和CD163的蛋白表达水平。在体外实验，PFS促进BMDMs增殖，PFS上调LPS刺激的巨噬细胞的Arg1，而抑制NOS2和磷酸化STAT3。免疫荧光结果表明PFS减轻LPS诱导巨噬细胞M1型极化和p65核转位。PFS抑制炎症因子表达，抑制MAPK信号相关蛋白表达。因此，PFS可能通过抑制Th反应和调节巨噬细胞极化来减轻噁唑酮诱导的AD的症状。 PFS可能用于治疗AD和相关的炎性皮肤病。.此外，我们还对广泛用于治疗中医风湿病和关节病的千斤拔进行实验研究。旨在探索千斤拔黄酮（FPF）对小鼠胶原诱导性关节炎（CIA）的骨侵蚀保护作用和抗炎作用机制。采用胶原诱导的C57BL/6小鼠关节炎模型。FPF灌胃显著抑制CIA小鼠的爪肿胀和关节炎评分。 FPF减少关节炎症浸润和血管翳形成，软骨破坏和破骨细胞浸润，以及MMP-9和组织蛋白酶K的表达。 FPF抑制CIA血浆抗CII抗体水平和炎性组织细胞因子和趋化因子的产生。 FPF处理抑制NF-κB的活化，下调CIA中 NF-κBp65和丝裂原活化蛋白激酶的磷酸化。另外，FPF通抑制激活蛋白-1和信号转导和转录激活因子3（STAT3）的激活。可见FPF可能是类风湿性关节炎的一种活性作用，FPF对CIA的预防作用可归因于预防骨质破坏，调节炎症介质和抑制NF-κB和MAPK信号通路。