基于Nox-hedgehog信号通路探讨畲药三叶青总黄酮抗肝纤维化药效物质基础及作用机制

Radix Tetrastigmehas, Traditional She Medicine (TSM), has the effect of clearing heat and detoxicating, promoting blood circulation and removing blood stasis, which is commonly used in the treatment of liver fibrosis (HF). Its curative effect is obvious, however, the material basis and mechanism remains unclear. Our previous studies have confirmed that the total flavonoids from Radix Tetrastigmehas has a clear effect on HF, which can inhibit the proliferation and induce the apoptosis of hepatic stellate cells. Recently, it has been found that Rac1, subunit of NOX, participates in regulating the Hedgehog pathway and promoting the activation of hepatic stellate cells, then leading the excessive proliferation and abnormal deposition of extracellular matrix (ECMs), which is the important pathological mechanism of HF development. In this paper, we propose a scientific hypothesis that the total flavonoids from Radix Tetrastigmehas can exert its anti HF effect by regulating the key cytokines of Nox-hedgehog signaling pathway and inhibiting the activation of HSCs. This project will track of active ingredients of anti HF based on the established microdialysis online technology in vivo and high content screening system of TCM method, in addition, combining the experiment of cell & whole animal level to study its effect of anti HF. Finally, we study its effect on Nox-hedgehog signaling pathway through CRISPR/Cas9, RT-PCR and Western Blot technology, revealing the material basis and mechanism of action of anti HF about the total flavonoids from Radix Tetrastigmae. Meanwhile, building the foundation for the development of novel anti HF drug.

畲药三叶青具有“清热解毒、活血化瘀”之功，临床常用于治疗肝纤维化（HF），疗效确切，优势明显，但物质基础及作用机制尚未明确。我们前期研究证实三叶青总黄酮对HF有明确的改善作用，可抑制肝星状细胞（HSCs）增殖并诱导其凋亡。新近研究发现NOX亚基Rac1调控Hedgehog通路，促进HSCs活化，致使细胞外基质（ECMs）过度增生和异常沉积，这是HF发展的重要病理机制。为此提出“三叶青总黄酮通过调控Nox-hedgehog通路关键因子抑制HSCs活化发挥抗HF作用”之科学假说。本项目建立基于在线活体组合微透析技术的高内涵中药筛选系统，追踪三叶青抗HF活性成分，从细胞-整体动物水平研究活性成分的抗HF作用，采取CRISPR/Cas9、RT-PCR、Western Blot等技术研究其对Nox-hedgehog通路的影响，揭示三叶青总黄酮抗HF物质基础及作用机制，为研发新型抗HF药物奠定基础。

中药三叶青具有“清热解毒、活血化瘀”之功，临床常用于治疗肝纤维化（HF），疗效确切，优势明显，但物质基础及作用机制尚未明确。本研究通过UPLC-QTOF MS/MS+GNPS及代谢组学从多维角度研究三叶青总黄酮（SYQTF）化学成分及其体内代谢物。体外共鉴定4种有机酸类化合物，2种酚酸及其衍生物，6种脂肪酸类化合物，29种其他类化合物，49种黄酮类化合物，且主要以山奈酚类为主；且浙江产地与其它产地SYQTF存在14种差异成分；体内血清共发现4种黄酮类化合物被吸收并转化为其相应代谢物，DESI肝脏组织成像可观察部分相关代谢产物。此外，SYQTF对四氯化碳（CCl4）诱导小鼠急性肝损伤具有肝保护作用，SYQTF干预后小鼠肝脏细胞轻度水肿，无明显坏死，脂质过氧化减轻，血清ALT、AST、LN和TNF-α显著下降。另外，SYQTF能抑制TGF-β1诱导HSC-T6细胞活化和增殖，促使其细胞周期停滞于G0/G1期，促进活化的HSC-T6细胞凋亡，并降低TNF-α、IL-6、Hyp、VEGF水平。与此同时，SYQTF干预组、SYQTF干预+Rac1慢病毒过表达组、Rac1慢病毒敲除组、SYQTF干预+Rac1慢病毒敲除组均可显著下调Rac1、Shh、Ptch1、Smo、Gli2的mRNA表达水平，上调Hhip的mRNA表达水平，并显著下调Rac1、Ptch1和Smo蛋白表达水平。以上结果均表明HF发病过程中Hedgehog (Hh) 通路激活依赖NOX亚基Rac1的过表达，且进一步揭示SYQTF干预Rac1调控Hh信号通路激活进而发挥抗HF作用分子机制，这为中药（畲药）三叶青临床应用及其抗肝纤维化新药研发提供重要理论支撑。