基于TGF-β/Smad 信号通路的莪术醋制抗肝纤维化代谢组学研究

The processing theory of traditional chinese medicine(TCM) is the characteristic of Chinese clinical medicine ,but the theory that guide decoction pieces application of safe and effective is short of modern scientific argument，thus it had become the bottle neck of Modernization of TCM. Hepatic fibrosis, the mutual pathologic change and inevitable stage in the development of most kinds of chronic liver diseases into hepatic cirrhosis and even hepatic cancer, was an unique reversible stage. The key mechanism in treatment of hepatic fibrosis are closely connect with signal channel and fibrinolytic system. To the problems mentioned above and the early stage research of our project, we introduce advanced technologies, such as LC-TOF/MS, polymerase chain reaction(PCR) and so on, to theoretical research field of processing innovatively. We discuss the regulation function of vinegar processed Curcumae Rhizoma to liver fibrosis key targets and its dynamic change of endogenous metabolism component, with hepatic stellate cells(HSC）, fibrinolytic system, transforming growth factor-beta(TGF - β)/Smad pathway and metabolomics, etc as the breakthrough point, through the overall identification and target analysis. We will further discuss the mechanism of anti-hepatic fibrosis effect of Curcumae Rhizoma processed with vinegar from molecular expression、signal transduction and metabonomics. To charify scientific connotation of that "Curcumae Rhizoma processed with vinegar played the anti-hepatic fibrosis role, through regulating the TGF-β/Smad signaling pathways, maintaining the system of urokinase-plasminogenactivator-matrix metalloproteinases- plasminogen activator inhibitor-1(uPA-MMPs-PAI-1) cascade reaction balance and changing endogenous metabolism of target components and specific metabolic pathways ". We aimed to provide a theoretical basis for Clinical Science utilization of vinegar processed Curcumae Rhizoma, annotate the traditional theory of processing with modern scientific language and provide a demonstration for modern research of processing theory.

中药炮制是中医临床辩证用药的特色，但指导饮片合理应用的炮制理论尚缺乏现代科学论据，成为制约中药现代化的瓶颈。莪术抗肝纤维化效应为临床验证，醋制后增强散瘀消积及抗肝纤维化作用，但醋制增效机制未见研究。肝纤维化是肝病发展为肝硬化或肝癌的必经阶段，是唯一可被逆转阶段。抗肝纤维化机制与信号通道及纤溶系统等关联密切。针对上述问题及前期研究基础，项目以HSC、信号通路、纤溶系统及代谢组学等为切入点，创新性地将LC-TOF/MS、PCR等前沿技术引入莪术醋制机制研究，探讨其对肝纤维化关键靶点的调控及代谢组分的动态变化。从分子表达、信号转导、代谢组学等环节系统研究，阐明莪术醋制是"通过调控TGF-β/Smad信号通路，维持uPA-MMPs-PAI-1级联反应系统平衡，改变机体内源性靶标代谢组分及特异代谢通路而增强抗肝纤维化作用"假说的科学内涵。为醋制莪术临床应用提供理论依据，也为中药炮制现代研究

莪术为临床抗肝纤维化中药，其醋制增效作用为临床所验证，但其增效机制尚未见研究。针对上述问题，本课题首次采用UPLC-TOF/MS方法对生、醋莪术饮片进行多成分定性分析，鉴定了其中20种差异成分。多元数据分析结果显示莪术经醋制后Curcumol、Curdione等16种成分含量有所升高，Curcumin、Demethoxycurcumin等4种成分含量降低，表明醋制对莪术化学成分产生明显影响。抗肝纤维化效应研究结果显示，莪术醋制能增强对肝细胞的保护作用；经生、醋莪术给药干预后的炎症因子表达均不同程度下降，且醋莪术效果更佳；莪术经醋制后能够抑制胶原等ECM的合成并促进其降解。此外，醋莪术能促进HSC-T6细胞凋亡，且增强对α-SMA和Procollagen I表达的抑制作用。对TGF-β/Smad 信号通路调控作用的研究结果表明，莪术抗肝纤维化的作用机制与减少TGF-β1的生成并抑制其受体表达，调控其作用底物Smads的表达，阻断TGF-β/Smad 信号通路的传导有关。对ECM降解酶系统调控作用研究结果表明，醋莪术经醋制后能够增强对PAI-1的调控作用，减少其对纤溶酶的抑制作用；同时减少TIMP-1的表达，减少其对MMP-2的抑制作用；调节MMP-2/TIMP-1的水平，维持ECM在生成与降解之间的平衡，从而达到抗肝纤维化的作用。代谢组学研究结果表明，莪术醋制前后对大鼠血清、尿液及肝组织代谢物谱具有显著影响，鉴定得LysoPE、LysoPC、Indolelactic acid，4-Hydroxyphenylpyruvic acid 等肝纤维化相关的潜在生物标记物，代谢通路分析表明莪术醋制增强抗肝纤维化效应的机制与CYP450等相关酶及TGF-β/Smad、NF-κB等信号通路密切相关。.综上所述，莪术醋制对其20种化学成分产生明显影响；效应研究表明，醋莪术能减少炎症因子的生成、炎性细胞浸润、有害因子对肝细胞的损害而保护肝细胞。莪术醋制后能增强对TGF-β/Smad信号通路的调控作用，阻断TGF-β1信号传导；抑制HSC的活化和增殖并诱导其凋亡，抑制ECM的生成并促其降解以及调控内源性代谢物水平从而影响相关通路表达而共同发挥抗肝纤维化作用。本课题从整体、组织、细胞分子水平揭示了莪术醋制抗肝纤维化的作用机制。为醋制莪术临床应用提供理论依据，也为中药炮制现代研究提供示范。