基于"主客交"理论的透邪通络法对肝纤维化作用的分子机制研究

Wu youke(one of the well-known warm disease doctors in Qing dynasty) proposed the theory "zhu ke jiao(pathogen invaded nutrient and blood aspects )" in the book named <wen yi lun (treatise on pestilence )> and created the famous formula named san jia san. Thereafter, Xue shengbai(warm disease doctor in Qing dynasty) carried forward his theory. This theory holds that because of the interaction between the pathogenic factor(ke) and the human physiological material(zhu), the human physiological material can induce disease to reflect the features of ke(pathogenic factor). Further more, the integration of zhu(physiological material) and ke(pathogenic factor) will accumulate in the blood solidly to caused stagnation of the collaterals, which finally leads to stubborn disease. This theory fits in with the main pathological process of activation of hepatic stellate cell and pathological intrahepatic capillary proliferation of hepatic fibrosis. A mass of clinical researches have found that the modification of San jia san can release hepatic fibrosis effectively. The research group has done some preliminary study on the functional mechanism of San jia san used to treat hepatic fibrosis. Therefore, this research is looking for solid evidences of the theory"zhu ke jiao(pathogen invaded nutrient and blood aspects )" .The approach is as following: we intervene the treatment which including the therapeutic method and herbs of rat model who suffered from immunological hepatic fibrosis, adopt theories and methods of molecular biology、immunology and proteomics to prove the effectiveness of this method used to treat hepatic fibrosis and to investigate the key cytokines and related signal paths of this method activating HSC(Hepatic Stellate Cell) and promoting angiogenesis, as well as to investigate the molecular mechanism of protein expression of sinusoidal endothelial cell. Finally, this research can rich the knowledge of the pathogenesis and therapeutic method of hepatic fibrosis. It has important theoretical value and clinical applying prospect.

吴又可《温疫论》首倡"主客交"之说并拟名方"三甲散"，其后薛生白承其说并加以发展。"主客交"病机理论认为：某些顽疾是由致病因素（客）与人体生理物质（主）相互作用，致生理物质产生致病性而具"客"的特性，即所谓"主客交浑"，其进一步"胶固于血脉"，导致"络脉凝瘀"而形成痼疾。此似与肝纤维化形成的主要病理环节- - -肝星状细胞激活及肝内病理性血管新生相契合。临床证实三甲散加减治疗肝纤维化确有良效，课题组前期对其作用机理进行了初步探索。由此，本研究拟通过对免疫性肝纤维化大鼠模型的治法方药干预，运用分子生物学、免疫学及蛋白组学等理论与方法，进一步验证透邪通络法治疗肝纤维化的有效性，考察其对HSC激活及其促血管新生过程中的关键细胞因子和相关信号通路，以及肝窦内皮细胞蛋白表达的影响，从治法机制角度，寻找"主客交"病机理论的实证依据。这对丰富中医对肝纤维化病机认识和治则治法，有重要的理论价值和临床应用前景。

肝纤维化疾病严重威胁人类健康，目前尚无理想的药物和治疗方法，中医药治疗肝纤维化显示出良好的应用前景，其理法方药有待进一步发掘。吴又可《温疫论》首倡“主客交”之说并拟名方“三甲散”，其后薛生白承其说并加以发展。“主客交”病机理论认为：某些顽疾是由致病因素（客）与人体生理物质（主）相互作用，致生理物质产生致病性而具“客”的特性，即所谓“主客交浑”，其进一步“胶固于血脉”，导致“络脉凝瘀”而形成痼疾。此似与肝纤维化形成的主要病理环节——肝星状细胞激活及肝内病理性血管新生相契合。临床证实三甲散加减治疗肝纤维化确有良效，课题组前期对其作用机理进行了初步探索。由此，在国家自然科学基金资助下，本研究通过对免疫性肝纤维化大鼠模型的治法方药干预，运用分子生物学、免疫学及蛋白组学等技术与方法，进一步验证了透邪通络法代表方加减三甲散能有效治疗肝纤维化，可降低肝组织HIF-1α、TGF-β1、Smad1、Smad3、VEGFR-2、PDGFB、ERK1 mRNA及蛋白的表达，抑制大鼠肝星状细胞激活和肝脏病理性血管新生从而阻断肝纤维化进程。同时，加减三甲散可升高肝组织P75蛋白含量，上调Bax蛋白表达，下调Bcl-2蛋白表达，促进HSC的凋亡从而抑制肝纤维化进展。其差异表达蛋白Cofilin2、ACSM3及NADH dehydrogenase可能是加减三甲散治疗肝纤维化的关键调控靶点之一。课题组已公开发表标记自然基金资助的学术论文8篇（中文核心4篇），其中获全国中医药博士生优秀论文二等奖1项，全国中医药博士生优秀论文指导老师奖1项，四川省中医药学会2016年学术年会优秀论文奖1项。培养硕士研究生4名，博士研究生2名，在本课题研究基础上申报并获得中国博士后科学基金项目1项，参加国内及国际学术交流4次，并作大会报告2次。研究结论部分揭示了“主客交”病机理论的现代科学内涵，从治法机制角度，为“主客交”病机理论提供了部分实证依据，丰富和深化了中医对肝纤维化的病机理论认识，拓展了透邪通络法的临床应用范围。