蒙药利德日-7君药成分苦参碱通过CRTC2抑制肥大细胞分泌IL-17A治疗银屑病的分子机理研究

Psoriasis was a common immune-mediated chronic inflammatory skin disease.And at present, the clinical treatment had high side-effect, and was easy to recur.IL-17A secreted by mast cells mediated inflammation in the pathogenesis of psoriasis, which was a hotspot of current drug development. Evaluation of the clinical curative effect of Mongolian medicine Li De Ri-7 showed it had very remarkable therapeutic effect. However, its effective components and the mechanism were unclear. The pre-experiment showed that the main components of Mongolian medicine Li De Ri-7 could inhibit mast cell secretion of IL-17A, and detected CRTC2 decreased at the same time. It has been suggested that CRTC2 could regulate IL-17A in Th17 cells, but there was no report on the function of regulating the IL-17A in mast cells. Therefore it was hypothesized that matrine by CRTC2 in inhibiting mast cell secretion of IL - 17A involved in the Mongolian medicine Li De Ri-7 in treatment of psoriasis. To verify the hypothesis, it is proposed: cell inflammation model and psoriasis animal model validated the function of matrine; The function of CRTC2 was validated by cell inflammation model; The molecular mechanism of matrine by CRTC2 in inhibiting mast cell secretion of IL-17A was explored by the cell model. Psoriasis clinical samples tested CRTC2 clinical significance would be discussed. The launch of this project would provide a scientific basis for clinical promotion of Mongolian medicine Li De Ri-7 in treatment of psoriasis.

银屑病是一种常见的免疫介导的慢性炎症性皮肤病，目前其临床治疗方法副作用多易复发。肥大细胞分泌IL-17A介导炎症参与银屑病的发病，是目前药物开发的热点。前期临床疗效评估中蒙药利德日-7具有很好的治疗效果。但其有效成分不清机理不明。预实验显示利德日-7主要成分苦参碱可以抑制肥大细胞分泌IL-17A，同时检测到CRTC2下降。有研究认为CRTC2可以在Th17细胞中调控IL-17A，但在肥大细胞中其是否具有调控IL-17A的功能未见报道。据此提出假设：苦参碱通过CRTC2抑制肥大细胞分泌IL-17A参与到利德日-7治疗银屑病中。为验证假设，拟：细胞炎症及动物类银屑病模型验证苦参碱的功能；细胞模型验证CRTC2的功能；细胞模型探讨苦参碱的分子机理为通过CRTC2抑制肥大细胞分泌IL-17A；银屑病临床样品检测探讨CRTC2临床意义。本项目的开展为蒙药利德日-7治疗银屑病的临床推广提供科学依据。

银屑病发病机制复杂，并在2014年被世界卫生组织认为是一种没有治愈方法的慢性非传染性疾病。课题负责人在长期的蒙医治疗银屑病的临床工作中发现利德日-7（LDR-7）对银屑病具有很好的治疗效果，但缺乏实验数据支持及作用机制研究。项目的主要研究内容及关键数据如下：（1）LC-ESI-MS/MS结果显示LDR-7水体液共含有160中化合物，主要包括氧化苦参碱、苦参碱、木犀草素等；体内实验表明，LDR-7能够显著降低咪喹莫特（IMQ）诱导的银屑病样小鼠皮肤PASI评分，HE染色结果同样证实LDR-7能够显著改善银屑病样小鼠皮损症状。（2）网络药理学对LDR-7治疗IMQ诱导银屑病样小鼠的机制进行预测，并通过分子生物学手段进行验证。网络药理学确定了与银屑病相关的活性成分对应的18个靶点；KEGG通路分析表明，IL-17信号通路、Toll样受体信号通路和JAK–STAT信号通路可能参与LDR-7治疗银屑病的过程。分子对接分析显示，5种生物活性化合物，苦参碱、胡椒碱、表小檗醇、苦苷III和木犀草素与相关靶蛋白具有良好的对接潜能。验证实验结果表明，LDR-7能够抑制银屑病样小鼠皮损组织中PCNA和Ki67蛋白表达，降低银屑病样小鼠血清中TNF-α和IL-17含量；LDR-7能够抑制TLR4/NF-κB、MAPK信号通路的过度激活，下调IL-17、IL-17R、ACT1、TRAF6、p-TAK1的表达水平，进而减少银屑病样小鼠皮肤组的过度炎症反应；降低p-JAK2及p-STAT3的磷酸化水平，缓解银屑病样小鼠皮肤组织细胞的过度增殖。（3）选取LDR-7君药成分苦参碱进行体外研究，结果表明苦参碱能够显著抑制TNF-α诱导的HacaT细胞的过度增殖，并能够抑制NF-κB p65的核转位，进而抑制NF-κB信号通路。基因芯片对苦参碱抑制TNF-α诱导的HacaT细胞的过度增殖的作用机制进行预测，结果表明其机制可能与细胞周期、MAPK信号通路、PPARα信号通路、TGF-β信号通路等相关。综上，项目研究结果表明，LDR-7能够通过抑制TLR4/NF-κB、MAPK、IL-17信号通路的过度激活，降低银屑病样小鼠血清相关炎症细胞因子含量，改善银屑病样小鼠皮肤的过度炎症，通过抑制JAK/STAT信号通路，抑制银屑病样小鼠皮肤组织中角质形成细胞的过度增殖，最终改善银屑病样小鼠的皮损症状。

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