基于EPCR脱落探讨大肠杆菌所致热毒血瘀证形成的分子机制及中药干预的研究

Noxious Heat and Blood Stasis Syndrome (NH-BS) is a type of syndromes in which noxious heat invade into the yingfen and xuefen syndrome. It includes varies of acute infectious diseases caused by bacteria and viruses in livestock. The different noxious heat invasion to yingfen and xuefen will lead to coagulation abnormalities and the same pathological changes such as bleeding and stasis. Endothelial protein C receptor (EPCR), which is a main member of protein C (PC) anticoagulant pathway, will shed from endothelial cell membrane as a soluble form when it was impared, which affects the effects of PC pathway. On the basis of previous experiments, we will research the molecular pathogenesis of E.coil-induced NH-BS, taking the EPCR and the signaling pathway of its shedding regulation as a starting point, using rat model of E.coil-induced NH-BS and aortic endothelial cell model inducing by the serum of the rat model, which will provide a new idea for studying the pathogenesis of NH-BS. In addition, we will study the effects of Xiang-Qi-Tang (XQT) on expression the EPCR protein and metalloproteinase ADAM17, as well as on PKC or MAPK signaling pathway, which will reveal the mechanisms of treating NH-BS using XQT, and provide experimental evidences and theoretical basis for the application of such traditional Chinese medicine in the veterinary clinic.

热毒血瘀证是热毒邪气侵入到营血分时出现的证候类型。多种由细菌、病毒引起的畜禽急性型传染病都属于此病证范畴。不同的"热毒"侵入到营血分都会使凝血功能异常导致出血、瘀血等相同的病理变化。血管内皮蛋白C受体（EPCR）是蛋白C（PC）抗凝途径的主要成员，受到损伤后从血管内皮细胞膜上脱落形成可溶性EPCR，严重影响PC途径的抗凝作用。本项目在前期试验基础上，以大肠杆菌所致热毒血瘀证大鼠模型和该病证大鼠血清损伤主动脉内皮细胞所造成的热毒血瘀证细胞模型为对象，以EPCR和调控其脱落的信号通路为切入点，探讨大肠杆菌所致热毒血瘀证形成的分子机制，为研究热毒血瘀证病机提供新的思路，同时研究香芪汤对EPCR和调控其脱落的金属蛋白酶ADAM17蛋白表达的影响以及对其上游PKC和MAPK信号途径的影响，揭示该方治疗热毒血瘀证的机制，为此类中药在兽医临床上的应用提供实验依据和理论基础。

热毒血瘀证是热毒邪气侵入到营血分时出现的证候类型。本项目在前期试验基础上，以大肠杆菌内毒素所致热毒血瘀证小鼠模型和内皮细胞EPCR脱落模型为对象，探讨大肠杆菌内毒素所致热毒血瘀证形成的分子机制，同时研究香芪汤对EPCR和调控其脱落的信号途径的影响。结果显示，小鼠腹腔注射5 mg/kg的LPS溶液，出现食欲下降，活动减少，眼结膜及耳部血管扩张充血，舌体呈鲜红色，眼睛分泌物增多，小便赤黄，大便臭秽等典型的热毒血瘀证的症状，运用western blot等方法研究发现在该病证模型中主动脉出现严重的EPCR脱落现象，并受到ADAM17-MAPK/PKC信号通路的调控，这可能是热毒血瘀证中凝血异常的主要分子机制。进一步研究发现，中药香芪汤及其单味药香附、黄芪、穿心莲能不同程度降低sAPP、TF、sEPCR的表达，提示香芪汤能不同程度保护血管内皮细胞，使PC抗凝途径、纤溶系统处于完整状态，减少高凝血液的发生率。同时，香芪汤能不同程度降低ADAM17、JNK、ERK、p38、PKCdelta的表达，表明香芪汤通过减少血管上MAPKs、PKC delta的表达，使ADAM17的活化受到抑制，进而使EPCR的脱落减少，起到保护血管内皮、增强机体抗凝的作用。为了进一步探讨香芪汤防治热毒血瘀证的分子机理，我们通过建立内皮细胞EPCR脱落模型，研究了香芪汤中三个主要有效成分α-香附酮、黄芪甲苷和穿心莲内酯调控EPCR的功效。结果发现用2μM PMA作用HUVECs 1小时可以最大程度的使EPCR脱落并且引起MAPK的磷酸化和PKC的转位以及磷酸化；α-香附酮通过抑制PKC的转位从而抑制TACE的表达和活性，从而抑制EPCR的脱落；黄芪甲苷通过抑制PKC和MAPK的磷酸化，抑制TACE的表达和活性，进而抑制EPCR的脱落；而穿心莲内酯只抑制MAPK的磷酸化和TACE的表达，从而抑制EPCR的脱落。综上，本研究的开展，初步阐明了热毒血瘀证的形成机制，为此类病证的防治提供新的药物作用靶点；阐明了香芪汤发挥治疗作用的机理，为此类中药的临床应用提供理论依据和实验基础。