CRF家族肽-受体系统在周围血管炎症的性别差异中的作用及其机制研究

TAO is more prevalent in male than female，whose mechanisms remain unknown. Previously, we found that CRF family peptide, UCN, promotes the development of TAO in rat model. Our recent new data show that under TAO condition, Male rats had significantly higher levels of serum UCN and more serious vasculitis than female ones. These results highly imply that UCN may play a significant role in the sex difference of TAO and sex hormones may function via UCN and its receptors.. Endothelial permeability is a major index for vasculitis, and UCN was found by us to increase lung vascular permeability. It was also reported that increased vascular permeability is associated with downregulation of VE-cadherin and beta-catenin binding to cadherin is a prequisite for maintaining cell-cell adhesion and hence permeability because this binding can protect cadherin from degradation. In addition, Beta-catenin is an important component of Wnt signaling pathway. When Beta-catenin is dissociated from cadherin, it is stablized in cytosol and then translocated into nucleus, interacts with TCF/LEF family, leading to gene transcription and some factor expression, like VEGF, which in turn functions as an inflammatory factor.. . This project is going to address the hypothesis that androgen plays a role in the sex difference in the incidence of TAO via influencing UCN and its CRF receptors and that UCN increases vascular permeability via disrupting Beta-catenin-caherin complex. The expected results will definitely contribute to the interpret of TAO mechanisms and the finding of drug targets for the treatment of TAO.

原发性血栓闭塞性脉管炎（TAO）绝大多数发生于男性，原因不明。我们发现，CRF家族肽对模型鼠TAO的发展起促进作用。新的预实验结果又显示，炎症时雄性与雌性相比，血液CRF肽UCN的水平显著提高，且雄性大鼠TAO更明显。这些结果高度提示雄激素可能通过影响UCN及其CRF受体而促进血管炎症发生发展。血管通透性增加是血管炎症的表现，我们发现UCN可增加肺血管通透性。而通透性增加与VE-cadherin下调相关，且β-catenin-cadherin复合物稳定是维持通透性的先决条件；β-catenin 又是Wnt信号通路的的重要成分。当β-catenin 从 VE-cadherin分离，会转位至胞核，与 TCF/LEF家族相互作用, 调节基因转录,进而影响VEGF等因子生成。本项目旨在阐明UCN在血管炎症性别差异中起到关键作用，其机制是通过使β-catenin-cadherin复合物分裂而实现的。

发现Ucn1下调钙粘蛋白VE-cadherin增加HUVECs通透性，阻断PKD和HSP27显著缓解该作用。Ucn1增加β-catenin Ser552位点磷酸化，促进cadherin-catenin解离、VE-cadherin泛素化，导致VE-cadherin表达下降。而β-catenin表达上升、转核，与T细胞因子/淋巴细胞增强子结合，激活β-catenin/Wnt信号通路，促进VEGF表达，进一步增加血管内皮细胞通透性；Ucn1激活cPLA2-NF-κB和cPLA2-COX-2-PGE2信号通路增加内皮ICAM-1的表达。Ucn1通过CRHR2增加ICAM-1及COX-2的表达，而增加ICAM-1表达依赖于COX酶的作用。Ucn1通过CRHR1和CRHR2增加cPLA2的表达及磷酸化。Ucn1增加NF-κB的磷酸化、促其进核，影响ICAM-1的转录。但NF-κB不参与Ucn1通过CRHR2对COX-2的表达调节作用。siRNA干扰cPLA2明显缓解COX-2的激活及PGE2的产生，亦阻止NF-κB和PKA-CREB通路激活；正常及炎症情况下，双氢睾酮DHT对内皮Ucn1表达的作用相反。DHT使Ucn1表达下降，该作用被雄激素受体(,AR)阻断剂消除；LPS存在时，DHT显著增加LPS诱导的Ucn1表达，且不受AR阻断剂的影响。DHT使AR表达上升、转核，抑制Ucn1表达。而有LPS时，DHT不影响AR表达和转运，但可增加TLR4表达和激活，该作用不依赖AR。DHT增加LPS诱导的p38MAPK,ERK1/2和NF-κB信号通路激活，这可能是DHT在炎症时促进内皮Ucn1表达的重要通路；发现Ucn1在大鼠月桂酸钠诱导的血管炎症模型性别差异中起重要作用。在股动脉注射月桂酸钠14天后，大鼠呈现典型的血管炎症症状、体征及股动脉的免疫组化表现。该模型存在明显的性别差异，雄性大鼠炎症情况明显重于雌性，血浆Ucn1、PGE2、sICAM-1浓度和股动脉Ucn1，COX-2和ICAM-1表达显著高于雌性。睾丸切除明显缓解雄性血管炎症症状，并伴随着血浆Ucn1表达下降。但切除后补充雌激素并没有进一步减轻炎症状态。在雌性大鼠中，卵巢切除后给予DHT明显增加Ucn1水平并加重血管炎症症状，补充雌激素无明显影响。给予兔来源抗Ucn1的免疫血清后几乎取消了大鼠血管炎症的性别差异。