白虎汤干预下家兔气分证证候相关蛋白互作机制

Qifen Syndrome is a typical representative of febrile disease in TCVM. It frequently appears in many animal infection or infectious diseases. It has an important guideline for animal disease control and drugs exploration to further study Qifen Syndrome. However, it is difficult to discover the scientific connotation of Qifen Syndrome through researching single-level experimental indicators. But the proteomics has higher feasibility to reveal the essence of the syndrome. We had researched liver function, tissue structure and gene express profile in Qifen Syndrome model and that was prevented by Bai-Hu-Tang in rabbits, and the results showed that the liver is significantly correlated to Qifen Syndrome. Therefore, we will use iTRAQ technology, which is based on tandem mass spectrometry, to research the differentially expressed proteins in animal liver tissue from rabbit with Qifen Syndrome induced by LPS and that of Bai-Hu-Tang-prevented animals, respectively. Then differentially expressed proteins will be analyzed through bioinformatics technology in order to find the proteins associated with Qifen Syndrome formation, and a network model of protein-protein interaction (PPI) will be established. The function of key proteins in the network will be analyzed by bioinformatics, and protein expression level will be determined by western blot and qRT-PCR, too. The same work will also be conducted in group of Bai-Hu-Tang prevention. Lastly, there will be a comparative analysis in two networks of PPI, and the syndrome essence and molecular mechanism of Qifen Syndrome formation will be demonstrated at protein level. The research result has important theoretical significance to further understand the scientific connotation of Qifen Syndrome.

气分证是典型的温病证候之一，出现在很多动物感染或传染性疾病中。气分证的深入研究对畜禽疾病防治和药物研发具有重要的指导价值。目前，单系统、单层次的实验指标研究已难以全面揭示证候的科学内涵，而蛋白组学研究对证候本质揭示的可行性更高。我们对家兔气分证的前期研究表明，肝脏的结构功能和基因表达谱的变化与气分证证候显著相关。为此，本项目拟采用基于串联质谱的iTRAQ技术，研究家兔气分证模型和经方白虎汤干预下动物肝组织中蛋白质组的差异表达，并结合生物信息学分析构建家兔气分证模型证候相关的蛋白互作网络数据模型，以及白虎汤干预下证候改善相关的蛋白互作网络模型，解析蛋白互作网络中的关键节点蛋白，并用免疫印迹和qRT-PCR进行分子表达验证。通过两组蛋白互作网络模型的比较分析，以方证对应的思路在蛋白水平阐明气分证证候本质和证候形成的分子机制。项目研究成果对深入理解气分证的现代科学内涵具有重要的理论意义。

本项目通过给家兔注射LPS建立气分证模型，并以白虎汤干预模型动物，从方证对应、以方测证的角度，利用蛋白组学技术首次在蛋白水平探究了气分证证候相关蛋白组学变化，以及白虎汤干预下证候相关蛋白表达的变化。同时，利用病理学、流式细胞术、酶联免疫吸附技术等常规技术手段研究了多个血液生物学指标变化。蛋白组学研究结果显示，气分证动物肝组织中核糖体通路中互作蛋白的表达变化引起该通路相关生物学功能变化，白虎汤干预后对核糖体通路变化有所改善，但不是其主要作用靶标，而以Coronin、F-actin、Rac和MHC I为关键节点蛋白的细胞吞噬作用通路是白虎汤治愈气分证的主要分子机制。血液学研究结果显示，气分证动物肝组织与肝功能都具有显著的病理损伤，血液中CD8+ T细胞数量降低，免疫球蛋白（IgG、IgM）含量显著高于正常动物，细胞因子（IL-2、-4、-5、-6、-10、TNF-α、TNF-β）含量也显著升高，血液内皮素、总补体CH50和C3a水平也显著升高，NO含量降低；白虎汤干预治疗气分证动物后，被损伤的肝组织结构与功能都有显著的改善和恢复，血液中CD+8 T细胞数量正常，免疫球蛋白（IgG、IgM）水平下降，部分细胞因子（IL-2、-6）含量降低，血液内皮素、总补体CH50和C3a含量显著下降，而拮抗内皮素的NO含量显著升高。结果表明，气分证时机体获得性免疫和先天免疫都被显著激活，机体免疫机能的过渡激活是气分证证候形成的主要原因，而白虎汤治愈气分证的主要机制与抑制免疫过渡激活相关。综合蛋白组学和多种医学指标分析表明，白虎汤通过促进细胞吞噬抗原、加工和交叉呈递抗原，进而激活细胞毒性T淋巴细胞以快速清除抗原，从而起到调控机体免疫过渡激活的作用。该项目在方证对应、以方测证的研究思路下，在蛋白水平阐明气分证证候本质和证候形成的分子机制，其研究成果对深入理解气分证的现代科学内涵具有重要的理论意义。